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Genetic Analysis of Photosynthetic Membrane Biogenesis in Rhodobacter sphaeroides
... (B) Amino acid sequence assigned from the pufx gene sequence of Rb. capsulatus (Youvan et al. 1984) and Rb. sphaeroides (Kiley et al. 1987) as well as chemically synthesized segments of these proteins (Parkes-Loach et al. 2001). PufX function (Francia et al. 2002). ...
The protein components of the reaction center (RC) and core light-harvesting (LH 1) complexes of photosynthetic bacteria have evolved to specifically, but non-covalently, bind bacteriochlorophyll (Bchl). The contribution to binding of specific structural elements in the protein and Bchl may be determined for the LH 1 complex because its subunit can be studied by reconstitution under equilibrium conditions. Important to the determination and utilization of such information is the characterization of the interacting molecular species. To aid in this characterization, a fluorescent probe molecule has been covalently attached to each of the LH 1 polypeptides. The fluorescent probes were selected for optimal absorption and emission properties in order to facilitate their unique excitation and to enable the detection of energy transfer to Bchl. Oregon Green 488 carboxylic acid and 7-diethylaminocoumarin-3-carboxylic acid seemed to fulfill these requirements. Each of these probes were utilized to derivatize the LH1 beta-polypeptide of Rhodobacter sphaeroides. It was demonstrated that the beta-polypeptides did not interact with each other in the absence of Bchl. When Bchl was present, the probe-labeled beta-polypeptides interacted with Bchl to form subunit-type complexes much as those formed with the native polypeptides. Energy transfer from the probe to Bchl occurred with a high efficiency. The alpha-polypeptide from LH 1 of Rb. sphaeroides and that from Rhodospirillum rubrum were also derivatized in the same manner. Since these polypeptides do not oligomerize in the absence of a beta-polypeptide, reversible binding of a single Bchl to a single polypeptide could be measured. Dissociation constants for complex formation were estimated. The relevance of these data to earlier studies of equilibria involving subunit complexes is discussed. Also involved in the photoreceptor complex of Rb. sphaeroides and Rhodobacter capsulatus is another protein referred to as PufX. Two large segments of this protein were chemically synthesized, one reproducing the amino acid sequence of the core segment predicted for Rb. sphaeroides PufX and the other reproducing the amino acid sequence predicted for the core segment of Rb. capsulatus PufX. Each polypeptide was covalently labeled with a fluorescent probe and tested for energy transfer to Bchl. Each was found to bind Bchl with an affinity similar to the affinity of the LH 1 polypeptides for Bchl. It is suggested that PufX binds Bchl and interacts with a Bchlcalpha-polypeptide component of LH 1 to truncate, or interupt, the LH 1 ring adjacent to the location of the Q(B) binding site of the RC.
... Working with Rhodopseudomonas viridis,Deisenhofer et al. (1985)crystallized the reaction center (RC). Shortly thereafter, George Fehers' and Jim Norris' laboratories (Allen et al. 1986;Schiffer and Norris 1993) crystallized the RC from R. sphaeroides for which a very robust gene exchange system had been developed (Nano et al. 1985;Davis et al. 1988;Suwanto and Kaplan 1992;Kaplan and Donohue 1993;Zeilstra-Ryalls et al. 1998b). C.N. Hunter and collaborators provided detailed spectroscopic analyses using a variety of cleverly constructed mutant strains of R. sphaeroides (Jones et al. 1992). ...
This minireview traces the photosynthesis genes, their structure, function and expression in Rhodobacter sphaeroides 2.4.1, as applied to our understanding of the inducible photosynthetic intracytoplasmic membrane system or ICM. This focus has represented the research interests of this laboratory from the late 1960s to the present. This opportunity has been used to highlight the contributions of students and postdoctorals to this research effort. The work described here took place in a much greater and much broader context than what can be conveyed here. The 'timeline' begins with a clear acknowledgment of the work of June Lascelles and William Sistrom, whose foresight intuitively recognized the necessity of a 'genetic' approach to the study of photosynthesis in R. sphaeroides. The 'timeline' concludes with the completed genome sequence of R. sphaeroides 2.4.1. However, it is hoped the reader will recognize this event as not just a new beginning, but also as another hallmark describing this continuum.
This minireview traces the photosynthesis genes, their structure, function and expression in Rhodobacter sphaeroides 2.4.1, as applied to our understanding of the inducible photosynthetic intracytoplasmic membrane system or ICM. This focus has represented the research interests of this laboratory from the late 1960s to the present. This opportunity has been used to highlight the contributions of students and postdoctorals to this research effort. The work described here took place in a much greater and much broader context than what can be conveyed here. The ‘timeline’ begins with a clear acknowledgment of the work of June Lascelles and William Sistrom, whose foresight intuitively recognized the necessity of a ‘genetic’ approach to the study of photosynthesis in R. sphaeroides. The ‘timeline’ concludes with the completed genome sequence of R. sphaeroides 2.4.1. However, it is hoped the reader will recognize this event as not just a new beginning, but also as another hallmark describing this continuum.
Under photosynthetic growth, purple non–sulfur photosynthetic bacterium Rhodobacter sphaeroides can use a diverse array of substrates for the source of carbon donor. Substrates such as acetate and succinate are most commonly used to study energetic and metabolic networks, especially in the production and consummation of NADPH during the citric acid cycle (TCA cycle) and ethylmalonyl–CoA pathway, respectively. Although the utilization of both substrate, the bacterium will grow at different growth rate and this also influence the biosynthesis of photosynthetic pigments as important components for overall photosynthesis. For this study, Rhodobacter sphaeroides strain 2.4.1, GA and G1C have been grown in acetate and succinate. Here, preliminary results on the evaluation the pigment ratio at different stages of the growth is reported, especially on the growth in succinate substrate.
The genome of the purple non-sulphur photosynthetic bacterium Rhodopseudomonas acidophila has been found to contain multiple copies of puc light-harvesting (LH2) peripheral antenna complex genes. Three wild-type isolates each exhibiting dissimilar peripheral antenna complex phenotypes in response to growth at reduced light intensity, were found to contain different numbers of these genes. Twenty-three puc cross-hybridising clones were isolated from a genomic library constructed from Rhodopseudomonas acidophila strain 7050; two of which were examined further; 2.6kb from one clone was sequenced and found to contain three β/α gene pairs designated puc
1BA, puc
2BA and puc
3BA. The putative translated polypeptides are very like, but not identical to those from B800–820 complexes and upstream sequence homologies suggests that this treble gene cluster has arisen through a relatively recent gene duplication event. From the other clone 0.6kb was sequenced and found to contain a further gene pair, puc
4BA, which is capable of encoding apoproteins for a B800-850-like complex. When the cells are grown at ‘high’ or ‘low’ light intensity Northern analyses showed that only puc
4BA is expressed under ‘high’ light conditions. Furthermore, pucBA mRNA transcripts were detected in all three species in the range 500–780 nt. In Rhodopseudomonas acidophila post-transcriptional regulatory mechanisms also play a role in determining the amount of peripheral antenna present in the intra-cytoplasmic membrane.
The process of the assembly of the pigment-protein complexes in R. sphaeroidesand R. capsulatus is only beginning to be revealed. The role of the RC-H polypep- tide as a component critical for the efficiency of association of the pigment-binding RC-L and -M polypeptides with the RC cofactors has been elucidated. The structural role of Bchl in RC complex stability is also being refined because of the availability of the RC crystal structure and the recently developed techniques of site-specific mutagenesis to alter critical Bchl-binding amino acids. The role of assembly proteins in RC assembly is, at present, established at the genetic level, but the gene products that are encoded by these genetic elements are yet to be characterized, and the order of events remains to be demonstrated. The amino acid similarity of both the LHI and LHII pigment-binding polypeptides and, as recently discovered, their putative assembly factors, indicates the close evolutionary relationship of these two light-harvesting complexes, both at the structural level and at the level of assembly. Likewise, the hypothetical existence of RC-specific assembly factors remains to be demonstrated, but in light of the evidence presented, their existence appears quite plausible. Finally, the orfQ gene product and its pivotal role in assembly of pigment-protein complexes and subsequent ICM development is clearly testable, and this system is ideal for future exploitation. The genetics and molecular genetic methodologies are established, the protein and cofactor components are readily available. Thus, researchers intent upon an understanding of membrane complex assembly processes have a wealth of potential at their disposal.
During photosynthesis light energy is converted into energy of chemical bonds through a series of electron and proton transfer reactions. Over the first ultrafast steps of photosynthesis that take place in the reaction center (RC) the quantum efficiency of the light energy transduction is nearly 100%. Compared to the plant and cyanobacterial photosystems, bacterial RCs are well studied and have relatively simple structure. Therefore they represent a useful model system both for manipulating of the electron transfer parameters to study detailed mechanisms of its separate steps as well as to investigate the common principles of the photosynthetic RC structure, function, and evolution. This review is focused on the research papers devoted to chemical and genetic modifications of the RCs of purple bacteria in order to study principles and mechanisms of their functioning. Investigations of the last two decades show that the maximal rates of the electron transfer reactions in the RC depend on a number of parameters. Chemical structure of the cofactors, distances between them, their relative orientation, and interactions to each other are of great importance for this process. By means of genetic and spectral methods, it was demonstrated that RC protein is also an essential factor affecting the efficiency of the photochemical charge separation. Finally, some of conservative water molecules found in RC not only contribute to stability of the protein structure, but are directly involved in the functioning of the complex.
We have been characterizing RNA polymerase holoenzymes from Rhodobacter sphaeroides. RNA polymerase purified from R. sphaeroides transcribed from promoters recognized by Escherichia coli E sigma 32 or E sigma 70 holoenzyme. Antisera to E. coli sigma 32 or sigma 70 indicated that related polypeptides of approximately 37 kDa (sigma 37) and 93 kDa (sigma 93), respectively, are present in this preparation. Transcription of sigma 32-dependent promoters was observed in a further fractionated R. sphaeroides holoenzyme containing the sigma 37 polypeptide, while a preparation enriched in sigma 93 transcribed sigma 70-dependent promoters. To demonstrate further that the sigma 93 polypeptide functions like E. coli sigma 70, we obtained an R. sphaeroides E sigma 93 holoenzyme capable of transcription from sigma 70-dependent promoters by combining sigma 93 with (i) an E sigma 37 fraction with diminished sigma 93 polypeptide content or (ii) E. coli core RNA polymerase. The generation of analogous DNase I footprints on the lacUV5 promoter by R. sphaeroides E sigma 93 and by E. coli E sigma 70 suggests that the overall structures of these two holoenzymes are similar. However, some differences in promoter specificity between R. sphaeroides E sigma 93 and E. coli E sigma 70 exist because transcription of an R. sphaeroides rRNA promoter was detected only with E sigma 93.
δ-Aminolevulinic acid dehydratase from Rhodopseudomonas spheroides, an enzyme which catalyzes the synthesis of the pyrrole, porphobilinogen, from 2 molecules of δ-aminolevulinic acid, has been purified 300- to 400-fold. The protein exhibits many of the characteristics of an allosteric enzyme. A plot of enzymatic activity versus substrate concentration yields a sigmoidal curve with an activity of about one-half that found in the presence of allosteric activators at high substrate concentrations. The allosteric effectors are monovalent cations, potassium, lithium, rubidium, and ammonium. A similar plot in the presence of these activators yields a hyperbolic saturation curve. Furthermore, the addition of K⁺ ions causes an association of the enzyme to form an equilibrium mixture of three enzymatically active species, a monomer, a dimer, and a trimer. The monomer, with a molecular weight of approximately 250,000, is the only species found on a sedimentation analysis in a sucrose gradient at very low concentrations of the above monovalent metallic cation. Sodium ions, on the other hand, do not fully activate the enzyme, as compared with the other alkali metal ions, whereas magnesium ions activate the enzyme to Vmax of about 80% of that found with K⁺, Li⁺, Rb⁺, and NH4⁺ ions. Manganese ions at low concentrations activate the enzyme but become inhibitory at higher concentrations.
The enzymatic activity is markedly inhibited by protoporphyrin and by hemin.
The mRNA transcripts of Rhodospirillum rubrum gene puh, coding for the H subunit of the photoreaction center, and of genes flanking puh were analyzed by blot hybridization. Open reading frame G115, upstream of structural gene puh, is transcribed as a 2.25-kilobase mRNA. Gene puh itself is transcribed as two mRNAs of 1118 and 1032 nucleotides. Mung bean nuclease protection analysis shows that the puh transcripts have different 5′ termini within open reading frame G115 and a unique rho-independent termination signal within open reading frame 12372. The lifetimes of the puh messages, as determined by an oxygen blockade of transcription, were 10 and 12 min for the large and small puh mRNAs, respectively. An expression vector carrying a chloramphenicol acetyltransferase gene was used to select promoters in DNA stretches upstream of the startpoints of each of these transcripts. Chloramphenicol resistance was expressed in Escherichia coli, using as a promoter a 179-nucleotide stretch upstream of the small mRNA startpoint but not from a 124-nucleotide stretch upstream of the large mRNA startpoint. The promoter for the small mRNA, designated Ppuh2, is thought to encompass in its −10 and −35 regions a σ⁷⁰-like RNA polymerase recognition sequence. The region upstream of the large message startpoint contains a sequence similar in its −12 and −24 regions to promoter sequences recognized by the σ⁶⁰ RNA polymerase holoenzyme. This is designated as promoter Ppuh1. Ppuh 1 is proposed to be strictly regulated by light intensity and by oxygen tension while Ppuh2 would be less sensitive to these parameters.
The determination of the structure of the Rps.
viridis reaction center at 3 A resolution1 has provided a great deal of information of direct relevance to the many other membrane proteins which are yet to be solved crystallographically. Two recent reviews2,3 have discussed putative structures for the quinol oxidizing complexes, and independently suggested a structure (the Cramer-Widger-Saraste-Wikstrom or CWSW structure) for the main subunit of the complexes (the cytochrome b subunit) in which the major structural elements are a set of nine membrane spanning helices. Two of these helices have a special role in providing pairs of histine residues to ligand the two b-type hemes of the complex. In this brief paper, we wish to review the structure of the cytochrome b subunit in the light of additional information from the reaction center structure, and from inhibitor resistant mutants, We will discuss first the structure of the catalytic site of the reaction center at which quinone is reduced, and our attempts to predict a tertiary structure for the analogous site on the photosystem II reaction center. We will examine the location of lesions giving rise to inhibitor resistance, and the information these provide about mechanism. We will then discuss the structure of the ubiquinol:cyt C2 oxidoreductases of R.
capsulatus and R.
sphaeroides, the location of the quinone reactive catalytic sites, and the role of inhibitor resistant mutants in elucidating the structure.
The puf operon of the purple nonsulfur photosynthetic bacterium, Rhodobacter sphaeroides, contains structural gene information for at least two functionally distinct bacteriochlorophyll-protein complexes (light harvesting and reaction center) which are present in a fixed ratio within the photosynthetic intracytoplasmic membrane. Two proximal genes (pufBA) specify subunits of a long wavelength absorbing (i.e., 875 nm) light harvesting complex which are present in the photosynthetic membrane in 15 fold excess relative to the reaction center subunits which are encoded by the pufLM genes. This review summarizes recent studies aimed at determining how expression of the R. sphaeroides puf operon region relates to the ratio of individual bacteriochlorophyll-protein complexes found within the photosynthetic membrane. These experiments indicate that puf operon expression may be regulated at the transcriptional, post-transcriptional, translation and post-translational levels. In addition, this review discusses the possible role(s) of newly identified loci upstream of pufB which may be involved in regulating either synthesis or assembly of individual bacteriochrlorophyll-protein complexes as well as the pufX gene, the most distal genetic element within the puf operon whose function is still unknown.
The topology of the cytochrome b subunit of the bc1 complex from Rhodobacter sphaeroides has been examined by generating gene fusions with alkaline phosphatase. Gene fusions were generated at random locations within the fbcB gene encoding the cytochrome b subunit. These fusion products were expressed in Escherichia coli and were screened for alkaline phosphatase activity on chromogenic plates. 33 in-frame fusions which showed activity were further characterized. The fusion junctions of all those fusions which had a high specific activity were clustered in three regions of the cytochrome b polypeptide, and thus these regions were tentatively assigned as being near the periplasmic surface. The data are consistent with a model containing eight transmembrane helices. In order to explore the validity of the gene fusion approach for a protein not normally expressed in E. coli, the topology of the L-subunit of the photosynthetic reaction center from R. sphaeroides was also explored using phoA gene fusions. A similar protocol was used as with the cytochrome b subunit. The gene fusions with high specific activity were shown to be in regions of the L-subunit polypeptide known to be at or near the periplasmic surface, as defined by the high resolution structure determined by X-ray crystallography. These data demonstrate the utility of this approach for determining membrane protein topology and extend potential applications to include at least some proteins not normally expressed in E. coli.
A genetic system has been developed for studying bacterial photosynthesis in the recently described nonsulfur purple photosynthetic bacterium Rhodospirillum centenum. Nonphotosynthetic mutants of R. centenum were obtained by enrichment for spontaneous mutations, by ethyl methanesulfonate mutagenesis coupled to penicillin selection on solid medium, and by Tn5 transposition mutagenesis with an IncP plasmid vector containing a temperature-sensitive origin of replication. In vivo and in vitro characterization of individual strains demonstrated that 38 strains contained mutations that blocked bacteriochlorophyll a biosynthesis at defined steps of the biosynthetic pathway. Collectively, these mutations were shown to block seven of eight steps of the pathway leading from protoporphyrin IX to bacteriochlorophyll a. Three mutants were isolated in which carotenoid biosynthesis was blocked early in the biosynthetic pathway; the mutants also exhibited pleiotropic effects on stability or assembly of the photosynthetic apparatus. Five mutants failed to assemble a functional reaction center complex, and seven mutants contained defects in electron transport as shown by an alteration in cytochromes. In addition, several regulatory mutants were isolated that acquired enhanced repression of bacteriochlorophyll in response to the presence of molecular oxygen. The phenotypes of these mutants are discussed in relation to those of similar mutants of Rhodobacter and other Rhodospirillum species of purple photosynthetic bacteria.
Cytochrome c2 is a periplasmic redox protein involved in both the aerobic and photosynthetic electron transport chains of Rhodobacter sphaeroides. The process of cytochrome c2 maturation has been analyzed in order to understand the protein sequences involved in attachment of the essential heme moiety to the cytochrome c2 polypeptide and localization of the protein to the periplasm. To accomplish this, five different translational fusions which differ only in the cytochrome c2 fusion junction were constructed between cytochrome c2 and the Escherichia coli periplasmic alkaline phosphatase. All five of the fusion proteins are exported to the periplasmic space. The four fusion proteins that contain the NH2-terminal site of covalent heme attachment to cytochrome c2 are substrates for heme binding, suggesting that the COOH-terminal region of the protein is not required for heme attachment. Three of these hybrids possess heme peroxidase activity, which indicates that they are functional as electron carriers. Biological activity is possessed by one hybrid protein constructed five amino acids before the cytochrome c2 COOH terminus, since synthesis of this protein restores photosynthetic growth to a photosynthetically incompetent cytochrome c2-deficient derivative of R. sphaeroides. Biochemical analysis of these hybrids has confirmed CycA polypeptide sequences sufficient for export of the protein (A. R. Varga and S. Kaplan, J. Bacteriol. 171:5830-5839, 1989), and it has allowed us to identify regions of the protein sufficient for covalent heme attachment, heme peroxidase activity, docking to membrane-bound redox partners, or the capability to function as an electron carrier.
Rhodobacter sphaeroides cytochrome c2 (cyt c2) is a periplasmic heme protein, encoded by cycA, that is required for photosynthetic growth and for one branch of the aerobic electron transport chain. cycA mRNA and cyt c2 are more abundant photosynthetically than aerobically. We report here that there are four cycA transcripts by high-resolution Northern (RNA) blot analysis, and we have mapped 10 5' ends by primer extension. Complementation of a cycA null mutant shows that there are at least two cycA promoters: one within 89 bp upstream of the translation initiation codon for a transcript beginning at -28, and at least one within 484 bp upstream for the remaining nine 5' ends. The 5' ends at -28 and -137 are more abundant in aerobically grown cells, while those at -38, -155, -250, and -300 are more abundant photosynthetically. DNA sequences with homology to the Escherichia coli sigma 70 consensus promoter sequence precede the 5' ends at -28 and -274, and there is weak homology upstream of the -82 and -250 ends.
The ubiquinol:cytochrome c2 oxidoreductase (bc1 complex) of Rhodobacter sphaeroides consists of four subunits. One of these subunits, cytochrome c1, is the site of interaction with cytochrome c2, a periplasmic protein. In addition, the sequences of the fbcC gene and of the cytochrome c1 subunit that it encodes suggest that the protein should be located on the periplasmic side of the cytoplasmic membrane and that it is anchored to the membrane by a single membrane-spanning alpha-helix located at the carboxyl-terminal end of the polypeptide. Site-directed mutagenesis of the fbcC gene was used to alter the codon for Gln228 to a stop codon. This results in the production of a truncated version of the cytochrome c1 subunit that lacks the membrane anchor at the carboxyl terminus. The bc1 complex fails to assemble properly as a result of this mutation, but the Rb. sphaeroides cells expressing the altered gene contain a water-soluble form of cytochrome c1 in the periplasm. The water-soluble cytochrome c1 was purified and characterized. The amino-terminal sequence is identical with that of the membrane-bound subunit, indicating the signal sequence is properly processed. High pressure liquid chromatography gel filtration chromatography indicates it is monomeric (28 kDa). The heme content and electrochemical properties are similar to those of the intact subunit within the complex. Flash-induced electron transfer kinetics measured using whole cells demonstrated that the water-soluble cytochrome c1 is competent as a reductant for cytochrome c2 within the periplasmic space. These data show that the isolated water-soluble cytochrome c1 retains many of the properties of the membrane-bound subunit of the bc1 complex and, therefore, will be useful for further structural and functional characterization.
The Rhodobacter sphaeroides gene encoding subunit IV of the cytochrome b-c1 complex (fbcQ) was cloned and sequenced. The fbcQ cistron is 372 base pairs long and encodes 124 amino acid residues. The molecular mass of subunit IV, deduced from the nucleotide sequence, is 14,384 Da. A hydropathy plot of the predicted amino acid sequence revealed only one transmembrane helix; it is near the C-terminal end. The 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]octyl)-1,4-benzoquinone ([3H]azido-Q)-labeled subunit IV was isolated from the [3H]-azido-Q-treated cytochrome b-c1 complex. A ubiquinone-binding peptide was obtained by digesting the labeled subunit IV with V8 protease followed by high performance liquid chromatography separation. Amino acid analysis and partial N-terminal sequencing of this ubiquinone-binding peptide revealed that it corresponded to residues 77-124 of subunit IV. Based on the hydropathy profile and predicted tendency to form alpha-helices and beta-sheets, we propose a structural model for subunit IV. In this model the ubiquinone-binding domain is located near the surface of the membrane.
Transcriptional expression of the puc operon in Rhodobacter sphaeroides 2.4.1 is dependent on the partial pressure of oxygen. By using transcriptional fusions in trans of a promoterless fragment derived from the aminoglycoside-3'-phosphotransferase gene of Tn903 to puc operon-specific DNA containing a 629-bp 5' cis-acting regulatory region involved in the expression of puc-specific mRNA, we selected Kmr colonies under aerobic conditions. Two broad classes of mutations, trans and cis, which are involved in O2 control of puc operon transcription, fall into several distinct phenotypic classes. The cis-acting regulatory mutations are characterized in detail elsewhere (J.K. Lee and S. Kaplan, J. Bacteriol. 174:1146-1157, 1992). Two trans-acting regulatory mutants, CL1a and T1a, which are B800-850- Car- and apparently B875-, respectively, were shown to derepress puc operon transcription in the presence of oxygen. The mutation giving rise to CL1a has been shown to act at the puc operon-specific cis-acting upstream regulatory region (-629 to -92). On the other hand, the mutation giving rise to T1a, identifying a second trans-acting regulatory factor(s), appears to act at both the upstream (-629 to -92) and the downstream (-92 to -1) regulatory regions of the puc operon as well as at the level(s) of bacteriochlorophyll and carotenoid biosyntheses, as revealed by the presence of the B800-850 complex under chemoheterotrophic growth conditions. Both the B800-850- Car- phenotype and the trans-acting effect on puc operon expression in mutant CL1a were complemented with a 2.2-kb DNA fragment located within the carotenoid gene cluster. Mutant T1a was complemented with a 7.0-kb EcoRI restriction fragment containing the puhA gene and its flanking DNA (6.3 kb) to restore expression of the B875 complex and to suppress the trans-acting effect resulting in the loss of 02 control. Under chemoheterotrophic conditions, mutant T1a was highly unstable, segregating into a PS- mutant designated T4.
Transcriptional expression of the puc operon in Rhodobacter sphaeroides is highly regulated by both oxygen and light. The approximately 600 bp of DNA upstream of the 5' ends of the two puc-specific transcripts encompasses two functionally separable cis-acting domains. The upstream regulatory region (URS) (-629 to -150) is responsible for enhanced transcriptional regulation of puc operon expression by oxygen and light. The more proximal upstream region (downstream regulatory region [DRS]), containing putative promoter(s), operator(s), and factor binding sites (-150 to -1), is involved in unenhanced transcriptional expression of the puc operon under aerobic and anaerobic conditions. Thus, the DRS shows normal derepression of puc operon expression when cells are shifted from aerobic to photosynthetic growth conditions in terms of percent change but does not show the potential range of expression that is only observed when elements of the URS are present. Because of these observations, we have made a distinction between anaerobic control (describing the shift) and oxygen control (describing the magnitude of derepression). Promoter(s) and/or activator function(s) of the puc operon is associated with a 35-bp DNA region between -92 and -57. Homologous sequences at -10 to -27 and -35 to -52 appear to involve additional regulatory elements: mutations at -12 (A to C) and -26 (G to A) result in partial derepression of puc operon expression under conditions of high aeration. Both point mutations require the upstream regulatory region (-629 to -150) to be present in cis for partial derepression of puc operon transcription under aerobic conditions. Immediately upstream of the promoter and/or activator region are overlapping consensus sequences for IHF (integratin host factor) and FNR (fumarate nitrate reductase) (-105 to -129). This region appears to be essential for enhanced expression of the puc operon. Thus, these two regulatory domains (URS and DRS) appear to involve approximately seven unique regulatory elements. In addition, the data reveal a direct interaction between the URS (-629 to -150) and the DRS (-150 to -1).
A 600-bp oriT-containing DNA fragment from the Rhodobacter sphaeroides 2.4.1 S factor (oriTs) (A. Suwanto and S. Kaplan, J. Bacteriol. 174:1124-1134, 1992) was shown to promote polarized chromosomal transfer when provided in cis. A Kmr-oriTs-sacR-sacB (KTS) DNA cassette was constructed by inserting oriTs-sacR-sacB into a pUTmini-Tn5 Km1 derivative. With this delivery system, KTS appeared to be randomly inserted into the genome of R. sphaeroides, generating mutant strains which also gained the ability to act as Hfr donors. An AseI site in the Kmr cartridge (from Tn903) and DraI and SnaBI sites in sacR-sacB (the levansucrase gene from Bacillus subtilis) were employed to localize the KTS insertion definitively by pulsed-field gel electrophoresis. The orientation of oriTs at the site of insertion was determined by Southern hybridization analysis. Interrupted mating experiments performed with some of the Hfr strains exhibited a gradient of marker transfer and further provided genetic evidence for the circularity and presence of two chromosomal linkage groups in this bacterium. The genetic and environmental conditions for optimized mating between R. sphaeroides strains were also defined. The results presented here and our physical map of the R. sphaeroides 2.4.1 genome are discussed in light of the presence of two chromosomes.
Rhodobacter sphaeroides 2.4.1 naturally harbors five cryptic endogenous plasmids (C. S. Fornari, M. Watkins, and S. Kaplan, Plasmid 11:39-47, 1984). The smallest plasmid (pRS241e), with a molecular size of 42 kb, was observed to be a self-transmissible plasmid which can transfer only to certain strains of R. sphaeroides. Transfer frequencies can be as high as 10(-2) to 10(-3) per donor under optimal mating conditions in liquid media in the absence of oxygen. pRS241e, designated the S factor, was also shown to possess a narrow host range, failing either to replicate or to be maintained in Escherichia coli, Agrobacterium tumefaciens, and Rhizobium meliloti. It was further revealed that one of the remaining four endogenous plasmids, pRS241d, was also transmissible at a frequency similar to that of the S. factor. As a cointegrate with pSUP203, S was maintained in E. coli, providing sufficient DNA from which a physical map of S could be constructed. Progressive subcloning of S-factor DNA, in conjunction with assays of plasmid transfer, led to the localization and identification of oriV (IncA), IncB, and the putative oriT locus. The DNA sequence of the 427 bp containing oriTs revealed topological similarity to other described oriT sequences, consisting of an A-T-rich DNA region, several direct and inverted repeats, and putative integration host factor (IHF)-binding sites, and was shown to be functional in promoting plasmid transfer.
We report the DNA sequence and mutational analysis of a novel cluster of six Bradyrhizobium japonicum genes of which at least three (designated cycV, cycW, and cycX) are essential for the formation of all cellular c-type cytochromes. Mutants having insertions in these genes were completely devoid of any soluble (periplasmic) or membrane-bound c-type cytochromes; even the apo form of cytochrome c1 was not detectable, neither in the membrane nor in the soluble fraction. As a consequence, the mutants had pleiotropic phenotypes such as defects in nitrate respiration, H2 oxidation, electron transport to cytochrome alpha alpha 3, and microaerobic respiration during symbiosis. A fourth open reading frame (ORF132) encoded a protein that might also be concerned with cytochrome c formation, but perhaps only indirectly. The other two open reading frames did not appear to function in this process. The predicted amino acid sequences of the cycW and cycX gene products suggested that these proteins were membrane-bound. The cycV gene product showed extensive similarity to the ATP-binding subunit of a superfamily of membrane-associated transport systems. The predicted ORF132 product was strikingly similar to bacterial thioredoxins and eukaryotic protein disulfide isomerase. Based on these findings it is possible that these proteins are members of a complex transport system involved in the biogenesis of all cytochromes c.
Cytochrome c2 (Mr 12,840) of the purple photosynthetic bacterium Rhodospirillum rubrum functions as a mobile electron carrier in the cyclic photosynthetic electron-transport system of this organism. It acts as the electron donor to photochemically oxidized reaction centres and is reduced in turn by electrons from the cytochrome bc1 complex. By using synthetic oligonucleotides based on the known amino acid sequence of the protein, the structural gene (cycA) has been identified and isolated. DNA sequence analysis indicates the presence of a typical prokaryotic 23-residue signal sequence, suggesting that the protein is synthesized as a precursor which is processed during its secretion into the periplasm. Evidence is presented for the production of assembled cytochrome c2 in Escherichia coli, but recombinants grow poorly and are unstable, suggesting toxicity of the gene product in this organism.
In Rhodobacter sphaeroides, cytochrome c2 (cyt c2) is a periplasmic redox protein required for photosynthetic electron transfer. cyt c2-deficient mutants created by replacing the gene encoding the apoprotein for cyt c2 (cycA) with a kanamycin resistance cartridge are photosynthetically incompetent. Spontaneous mutations that suppress this photosynthesis deficiency (spd mutants) arise at a frequency of 1 to 10 in 10(7). We analyzed the cytochrome content of several spd mutants spectroscopically and by heme peroxidase assays. These suppressors lacked detectable cyt c2, but they contained a new soluble cytochrome which was designated isocytochrome c2 (isocyt c2) that was not detectable in either cycA+ or cycA mutant cells. When spd mutants were grown photosynthetically, isocyt c2 was present at approximately 20 to 40% of the level of cyt c2 found in photosynthetically grown wild type cells, and it was found in the periplasm with cytochromes c' and c554. These spd mutants also had several other pleiotropic phenotypes. Although photosynthetic growth rates of the spd mutants were comparable to those of wild-type strains at all light intensities tested, they contained elevated levels of B800-850 pigment-protein complexes. Several spd mutants contained detectable amounts of isocyt c2 under aerobic conditions. Finally, heme peroxidase assays indicated that, under anaerobic conditions, the spd mutants may contain another new cytochrome in addition to isocyt c2. These pleiotropic phenotypes, the frequency at which the spd mutants arise, and the fact that a frameshift mutagen is very effective in generating the spd phenotype suggest that some spd mutants contain a mutation in loci which regulate cytochrome synthesis.
We have used genetic and biochemical techniques to study carotenoid biosynthesis (crt) mutants of Rhodobacter capsulatus, a purple non-sulfur photosynthetic bacterium. All nine identified crt genes are located within the 46-kilobase pair photosynthesis gene cluster, and eight of the crt genes form a subcluster. We have studied the operon structure of the crt gene cluster using transposon Tn5.7 mutants. The Tn5.7 insertion sites in 10 mutants have been mapped to high resolution (25-267 base pairs) by Southern hybridization. Two insertions each map within the coding regions of the crtA, crtC, crtE, and crtF genes, and one insertion lies within the crtI gene. The insertion in crtI is not polar on the downstream crtB gene, suggesting that crtI and crtB may form two separate operons. Another insertion located in the 5' noncoding region between the divergent crtA and crtI genes has no effect on wild-type pigmentation and apparently lies between the promoters for these operons. A Tn5.7 mutation in the 3' region of crtA yields a bacteriochlorophyll-minus phenotype, while a 5' insertion affects only carotenoid biosynthesis. Regulatory signals for transcription of a downstream operon required for bacteriochlorophyll biosynthesis may thus overlap the coding region of crtA. We also present the first evidence for the functions of the crtB, crtE, and crtJ gene products using a new in vitro assay for the incorporation of [14C]isopentenyl pyrophosphate into carotenoid precursors and phytoene in cell-free extracts. Extracts from a crtE mutant accumulate [14C]prephytoene pyrophosphate, while those from crtB and crtJ mutants accumulate [14C]geranylgeranyl pyrophosphate. We therefore propose that CrtE is the phytoene synthetase and that CrtB, and possibly CrtJ, are components of the prephytoene pyrophosphate synthetase.
Purple non-sulfur photosynthetic bacteria such as Rhodobacter sphaeroides can grow by aerobic respiration, by photosynthesis under anaerobic conditions in the light, or by anaerobic respiration in the dark if electron acceptors such as dimethylsulfoxide (DMSO), trimethylamine-Noxide or nitrous oxide are present (1) (R. sphaeroides sp. denitrificans can also use nitrate or nitrite; 2,3). Given this metabolic and energetic versatility, it is not surprising that this Gram-negative bacterium contains many cytochromes whose synthesis can be environmentally regulated.
A cytochrome bc
1-complex of Rs. rubrum was isolated and the three subunits were purified to homogeneity. The N-terminal amino acid sequence of the purified subunits was determined by automatic Edman degradation. The pet genes of Rhodospirillum rubrum coding for the three subunits of the cytochrome bc
1-complex were isolated from a genomic library of Rs. rubrum using oligonucleotides specific for conserved regions of the subunits from other organisms and a heterologous probe derived from the genes for the complex of Rb. capsulatus. The complete nucleotide sequence of a 5500 by SalI/SphI fragment is described which includes the pet genes and three additional unidentified open reading frames. The N-terminal amino acid sequence of the isolated subunits was used for the identification of the three genes. The genes encoding the subunits are organized as follows: Rieske protein, cytochrome b, cytochrome c
1. Comparison of the N-terminal protein sequences with the protein sequences deduced from the nucleotide sequence showed that only cytochrome c
1 is processed during transport and assembly of the three subunits of the complex. Only the N-terminal methionine of the Rieske protein is cleaved off. The similarity of the deduced amino acid sequence of the three subunits to the corresponding subunits of other organisms is described and implications for structural features of the subunits are discussed.
The cytochrome b subunit of the bc1 complex contains two cytochrome components, cytochrome b(H) and cytochrome b(L) Sequence comparisons of this polypeptide from a number of organisms have revealed four invariant histidines which have been postulated to be the heme ligands for the two protoheme IX prosthetic groups. In Rhodobacter sphaeroides, these correspond to His97, His111, Hisl98, and His212. In this paper, the results of amino acid substitutions at each of these positions are reported. Replacement of His97 by either Asp or Asn and of Hisl98 by Asn or Tyr resulted in loss of both cytochrome components. However, His111Asn, His111Asp, and His2l2Asp all resulted in the selective loss of cytochrome b(H) and the retention of cytochrome b(L). Furthermore, flash kinetics studies show that the myxothiazol-sensitive quinol oxidase (Q(z)) site associated with cytochrome b(L) is still functional. These data support the assignment of the axial ligands to cytochrome b(H) (His111 and His212) and cytochrome b(L) (His97 and His198). This pairing is consistent with current models of the cytochrome b subunit with eight transmembrane a-helices.
The primary reaction of photosynthesis is light-driven charge
separation, carried out in reaction centres, which are complexes of
integral membrane proteins and cofactors. The recent determination of
the crystal structures of the reaction centres of two photosynthetic
bacteria provides a basis for a quantitative understanding of the
primary electron transfer processes of photosynthesis.
Rhodobacter sphaeroides mutants lacking cytochrome c2 (cyt c2) have been constructed by site-specific recombination between the wild-type genomic cyt c2 structural gene (cycA) and a suicide plasmid containing a defective cyc operon where deletion of cycA sequences was accompanied by insertion of a KnR gene. Southern blot analysis confirmed that the wild-type cyc operon was exchanged for the inactivated cycA gene, presumably by double-reciprocal recombination. Spectroscopic and immunochemical mea- surements, together with genetic complementation, established that the inability of these mutants to grow under photosynthetic conditions was due to the lack of cyt c2. The cyt c2 deficient strains reduced pho- tooxidized reaction center complexes approximately 4 orders of magnitude more slowly than the parent strain. The phenotype and characteristics of these mutants were restored when a wild-type cyc operon was introduced on a stable low copy number plasmid. These experiments provide the first genetic evidence for the obligatory role of cyt c2 in wild-type cyclic photosynthetic electron transport in R. sphaeroides. We have also observed that the R. sphaeroides cyt c2 deficient strains spontaneously gave rise to photosynthetically competent pseudorevertants at a frequency which suggests that the cyt c2 independent photosynthetic electron transport which suppresses the phenotype of the cyt c2 deficient strains was the result of a single mutation elsewhere in the genome.
An azidoubiquinone derivative, 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyloctyl)-1,4-benzoquinone, was used to study the ubiquinone-protein interaction and to identify ubiqumone-binding proteins in photosynthetic bacterial cytochrome b-c1 complex. When isolated Rhodobacter sphaeroides cytochrome b-c1 complex is incubated with a 50-fold molar excess of the azidoubiquinone derivative in the dark, no loss of activity is observed. Photolysis of this azidoubiquinone-treated sample for 5 min at 0 °C causes a 50% decrease of ubiquinol-cytochrome c reductase activity. When the photolyzed [3H]azidoubiquinone-treated R. sphaeroides cytochrome b-c1 complex is subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, after removal of non-protein-linked azidoubiquinone by organic solvent extraction, followed by analysis of the radioactivity distribution among subunits of the complex, cytochrome b (Mr 43K) and a Mr 12K protein are heavily labeled, suggesting that these two proteins are the ubiquinone-binding site in this complex. The amount of radioactivity in both proteins is increased when the complex is subjected to phospholipase A2 digestion prior to photolysis with the azidoubiquinone derivative. Pretreatment of R. sphaeroides cytochrome b-c1 complex with 2-heptyl-4-hydroxyquinoline ,N-oxide has little effect on the distribution of radioactivity among subunits of the cytochrome b-c1 complex. Pretreatment of the cytochrome b-c1 complex with 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole, myxothiazoi, or antimycin increases slightly the amount of radioactivity in cytochrome b. These results suggest that the active site of these inhibitors is not the same as the Q-binding site.
Photosynthetic electron flow from the cytochrome bc1 complex to the reaction center has been studied in a strain of Rhodopseudomonas capsulata which has had the gene for cytochrome c2 deleted from its genome. Previously, cytochrome c2 was thought to be essential for electron flow between these two complexes, but we find this not to be the case in R. capsulata. Indeed, in this organism it seems likely that cytochrome c1 is able rapidly (t1/2 < 100 μs) to transfer electrons directly to the reaction center. However, this reaction is incomplete; only some 20% of the reaction centers are reduced in this way. In the wild type, a further 20% is rapidly reduced by cytochrome c2, but the remaining reaction centers are reduced rather more slowly by an as yet unidentified route that may involve cytochrome c2 shuttling between complexes. The deletion of the cytochrome c2 gene allows a determination of the oxidation-reduction midpoint potential of cytochrome c1 in the absence of cytochrome c2: cytochrome c1 has an Em7 of 345 mV. Furthermore, the finding that a phase of the electrogenic carotenoid bandshift accompanies the oxidation of cytochrome c1 in the absence of c2 indicates that the heme of cytochrome c1 must be near the inner aqueous-membrane interface of the chromatophore.
Reaction centers (RCs) from the photosynthetic bacterium Rhodopseudomonas sphaeroides are composed of three subunits (designated L, M, and H) plus cofactors. An active and stable LM complex containing approximately one Fe per LM was obtained from these RCs by dissociating the H subunit with the chaotropic agent LiClO4 in the presence of mild detergents such as sodium cholate. The LM was characterized and compared with native RCs in a variety of assays. RCs reconstituted from LM and H were indistinguishable from native RCs in all assays performed. Comparison of LM with native RCs showed that removal of H resulted in little or no change in (1) primary photochemical activity (i.e., bleaching of the absorption at 865 nm upon illumination), (2) Fe and quinone content, (3) narrow and broad EPR signals due to (BChl)2+ and the QA-Fe2+complex, respectively, (4) kinetics of charge recombination between QA- and (BChl)2+, and (5) kinetics of oxidation of ferrocytochrome c by (BChl)2+. These results show that the H subunit has little or no effect on the binding sites of Fe and cytochrome and that both primary and secondary quinone acceptors, QA and QB, bind to LM. However, electron transfer involving QA and QB was dramatically affected by the absence of the H subunit. Comparison of LM with native and reconstituted RCs showed that removal of H resulted in (1) reduction in the rate of electron transfer from QA- to QB by a factor of 102-103, (2) reduction in the stability of the semiquinone anions QA- and QB- by a factor of 102-103, (3) elimination of the oscillations of the absorption of semiquinone in response to successive flashes of light, (4) reduction in the affinity of Q-10 for the QB site by a factor of ∼ 10, (5) reduction in sensitivity to inhibitors of electron transfer (e.g., o-phenanthroline and terbutryn) by a factor of ∼102, and (6) reduction in stability to denaturation by detergents. These results suggest that the H subunit plays a major role in defining the environment of the QB site.
Two kinds of mutants of Rhodopseudomonas sphaeroides that should be useful in extending genetic analysis of this organism have been isolated. One is deficient in recombination and has been used to isolate derivatives of the plasmid R 68.45 which incorporate chromosomal genes of R. sphaeroides. The other is apparently defective in a DNA restriction enzyme; transfer of plasmid borne chromosomal genes of R. sphaeroides from Escherichia coli back to R. sphaeroides is greatly enhanced in these mutants.
Structural changes association with the intracytoplasmic membrane during the cell cycle of the photosynthetic bacterium Rhodopseudomonas sphaeroides have been studied by freeze-fracture electron microscopy. The isolated intracytoplasmic membrane vesicles, chromatophores, were fused in order to obtain large fracture faces, allowing more precise measurements and statistical analysis of both intramembrane particle density and size determinations. The intramembrane particle density of the protoplasmic face (PF) of the intracytoplasmic membrane, (from 4970 to 8290/μm2), was shown to be a linear function of the protein/ phospholipid ratio (from 2.5 to 5.1, w/w) of the intracytoplasmic membrane. Under constant light intensity, both the average particle size and particle size distribution remained unchanged during the cell cycle. These results provide the structural basis for the earlier reported cell-cycle-specific variations in both protein/ phospholipid ratio and alternation in phospholipid structure of the intracytoplasmic membrane of R. sphaeroides during photosynthetic growth. The average particle diameter in the PF face of the intracytoplasmic membrane was 8.25, 9.08 and 9.75 nm at incident light intensities of 4000, 500 and 30 ft · cd, respectively. When chromatophores were fused with small, unilamellar liposomes, the intramembrane particle density decreased as input liposome phospholipid increased, whereas the particle size remained constant and particle distribution became random.
Reaction centers from wild-type Rhodobacter sphaeroides (formerly called Rhodopseudomonas sphaeroides) were separated into two components: the LM complex and H subunit. LM was isolated after brief treatment of reaction centers with SDS by affinity chromatography with cytochrome c as ligand. A stable H preparation was obtained after dissociation of reaction centers with lithium perchlorate. LM was depleted of the transition metal, Mn, which interacts with QA and QB in native reaction centers. It retained only 30% of primary photochemistry which could be restored to 50–80% by addition of Q6, Q10 or other quinones. A stable semiquinone radical Q−A could be flash-induced in LM. Its absorption properties are similar to those of Q−A in native reaction centers. The quantum yield of photochemistry in an LM unit reconstituted with Q6 is the same as in intact reaction center and in LM in the presence of H. This result was confirmed by the rapid electron-transfer rate between I− and QA in LM + H (τ ≈ 0.45 ns). Ubiquinone in LM incubated with H becomes tightly bound at the QA site. Flash production of a Q2− species was not detected in LM and LM + H. We conclude that the depletion of the reaction center both of the H subunit and of the metal does not necessarily lower the quantum yield of the primary reaction or greatly modify the rate of electron transfer from I− to QA. These results contrast with observations of others that seemed to demonstrate that the metal is essential for high-rate electron transfer between I− and QA (Debus, R.J., Feher, G. and Okamura, M.Y. (1986) Biochemistry 25, 2276–2287). In our experiments, secondary electron transfer to QB was not restored in LM + H, unlike in reconstitution experiments reported with R26 Rb. sphaeroides reaction centers (Debus, R.J., Feher, G. and Okamura, M.Y. (1985) Biochemistry 24, 2488–2500). Apparently, interactions between H and LM were too weak for restoring QB activity.
A new method for in vitro insertional mutagenesis of genes cloned in Escherichia coli is presented. This simple procedure combines the advantages of in vitro DNA linker mutagenesis with those of in vivo transposition mutagenesis. It makes use of the Ω fragment, a 2.0-kb DNA segment consisting of an antibiotic resistance gene (the Smr/Spcr gene of the R100.1 plasmid) flanked by short inverted repeats carrying transcription and translation termination signals and synthetic polylinkers. The Ω fragment is inserted into a linearized plasmid by in vitro ligation, and the recombinant DNA molecules are selected by their resistance to streptomycin and spectinomycin. The Ω fragment terminates RNA and protein synthesis prematurely, thus allowing the definition and mapping of both transcription and translation units. Because of the symmetrical structure of Ω, the same effect is obtained with insertions in either orientation. The antibiotic resistance gene can be subsequently excised from the mutated molecules, leaving behind its flanking restriction site(s).
We describe several new vectors for the construction of operon and protein fusions to the Escherichia colilacZ gene. In vitro constructions utilize multicopy plasmids containing suitable cloning sites located between upstream transcription terminators and downstream lac operon segments whose lacZ genes retain or lack translational start signals. Single-copy λ prophage versions of multicopy constructs can be made genetically, without in vitro manipulation. The new vectors, both single and multicopy, are improved in that they have very low levels of background lac gene expression, which makes possible the easy detection and accurate quantitation of very weak transcriptional and translational signals. These vectors were developed for analysis of the expression of IS 10's transposase gene, which is transcribed less than once per generation, and whose transcripts are translated on average less than once each. Both single and multicopy constructs can also be used to select mutations affecting fusion expression, and mutations isolated in single-copy constructs can be crossed genetically back onto multicopy plasmids for further analysis.
Light-harvesting mutants of Rhodopseudomonas sphaeroides lacking either the B800–850 complex or the B875 complex have been characterized by their absorption spectra in the visible and near-infrared region, and by their ability to transfer energy from the light-harvesting complexes to the reaction center. A new method of measuring the relative efficiency of energy transfer from the light-harvesting complexes to the reaction center is described. The B875− mutant had absorption maxima in the near-infrared at 800 and 849 nm with no evidence of an 875-nm shoulder. The efficiency of energy transfer from the light-harvesting complexes to the reaction center in the B875− mutant was 24% of the value measured for the wild-type strain and the B800–850− mutant. Yet, despite the fact that the efficiency of energy transfer for the B800–850− mutant and the wild-type strain were the same, there was a large difference in their photosynthetic unit size. These results are discussed in the context of a model in which light energy captured by the B800–850 complexes is transferred through the B875 complexes to the reaction center.
Rhodopseudomonas spheroides was grown aerobically in the dark in continuous culture yielding cells with a low bacteriochlorophyll content. The supernatant obtained after cell breakage contained a mixture of cytochromes, apparently of the c-type; these were resolved by spectroscopy at 77°K into three components. The positions of the α, β and γ bands in reduced minus oxidised difference spectra at 77°K were 547, 520 and 417 nm; 549, 520 and 420 nm and 551, 521 and 421 nm, respectively. The 547-nm cytochrome was not readily reduced by ascorbate/tetramethyl-p-phenylene diamine (TMPD) and the 551-nm cytochrome was autoxidisable. Both the 547- and the 549-nm cytochrome were found in the particulate fraction and both were reduced by NADH or succinate in the aerobic steady state and became more oxidised in the presence of antimycin or 2-heptyl-4-hydroxyquinone-N-oxide (HQNO). CO difference spectra of the soluble fraction indicated that it also contained cytochromoid c; maxima were obtained at 569, 539 and 416 nm.
(1) The role of the ubiquinone pool in the reactions of the cyclic electron-transfer chain has been investigated by observing the effects of reduction of the ubiquinone pool on the kinetics and extent of the cytochrome and electrochromic carotenoid absorbance changes following flash illumination. (2) In the presence of antimycin, flash-induced reduction of cytochrome b-561 is dependent on a coupled oxidation of ubiquinol. The ubiquinol oxidase site of the ubiquinol:cytochrome c(2) oxidoreductase catalyses a concerted reaction in which one electron is transferred to a high-potential chain containing cytochromes c(1) and c(2), the Rieske-type iron-sulfur center, and the reaction center primary donor, and a second electron is transferred to a low-potential chain containing cytochromes b-566 and b-561. (3) The rate of reduction of cytochrome b-561 in the presence of antimycin has been shown to reflect the rate of turnover of the ubiquinol oxidase site. This diagnostic feature has been used to measure the dependence of the kinetics of the site on the ubiquinol concentration. Over a limited range of concentration (0-3 mol ubiquinol/mol cytochrome b-561), the kinetics showed a second-order process, first order with respect to ubiquinol from the pool. At higher ubiquinol concentrations, other processes became rate determining, so that above approx. 25 mol ubiquinol/mol cytochrome b-561, no further increase in rate was seen. (4) The kinetics and extents of cytochrome b-561 reduction following a flash in the presence of antimycin, and of the antimycin-sensitive reduction of cytochrome c(1) and c(2), and the slow phase of the carotenoid change, have been measured as a function of redox potential over a wide range. The initial rate for all these processes increased on reduction of the suspension over the range between 180 and 100 mV (pH 7). The increase in rate occurred as the concentration of ubiquinol in the pool increased on reduction, and could be accounted for in terms of the increased rate of ubiquinol oxidation. It is not necessary to postulate the presence of a tightly bound quinone at this site with altered redox properties, as has been previously assumed. (5) The antimycin-sensitive reactions reflect the turnover of a second catalytic site of the complex, at which cytochrome b-561 is oxidized in an electrogenic reaction. We propose that ubiquinone is reduced at this site with a mechanism similar to that of the two-electron gate of the reaction center. We suggest that antimycin binds at this site, and displaces the quinone species so that all reactions at the site are inhibited. (6) In coupled chromatophores, the turnover of the ubiquinone reductase site can be measured by the antimycin-sensitive slow phase of the electrochromic carotenoid change. At redox potentials higher than 180 mV, where the pool is completely oxidized, the maximal extent of the slow phase is half that at 140 mV, where the pool contains approx. 1 mol ubiquinone/mol cytochrome b-561 before the flash. At both potentials, cytochrome b-561 became completely reduced following one flash in the presence of antimycin. The results are interpreted as showing that at potentials higher than 180 mV, ubiquinol stoichiometric with cytochrome b-561 reaches the complex from the reaction center. The increased extent of the carotenoid change, when one extra ubiquinol is available in the pool, is interpreted as showing that the ubiquinol oxidase site turns over twice, and the ubiquinone reductase sites turns over once, for a complete turnover of the ubiquinol:cytochrome c(2) oxidoreductase complex, and the net oxidation of one ubiquinol/complex. (7) The antimycin-sensitive reduction of cytochrome c(1) and c(2) is shown to reflect the second turnover of the ubiquinol oxidase site. (8) We suggest that, in the presence of antimycin, the ubiquinol oxidase site reaches a quasi equilibrium with ubiquinol from the pool and the high- and low-potential chains, and that the equilibrium constant of the reaction catalysed constrains the site to the single turnover under most conditions. (9) The results are discussed in the context of a detailed mechanism. The modified Q-cycle proposed is described by physicochemical parameters which account well for the results reported.
Monospecific antibodies have been prepared against cytochrome c2 from Rhodopseudomonas spheroides and Rhodopseudomonas capsulata, and against cytochrome c' from Rps. capsulata. These antibodies precipitated their respective antigens, but did not cross react with a wide range of procaryotic or eucaryotic cytochromes, or with other bacterial proteins. The cytochromes produced during aerobic growth were immunologically indistinguishable from those produced during photosynthetic growth. Cytochrome c2 is located in vivo in the periplasmic space between the cell was and the cell membrane, and when chromatophores are prepared from whole cells the cytochrome becomes trapped inside these vesicles. The implications of these results to energy coupling in the photosynthetic bacteria are discussed.
In Rhodobacter sphaeroides, mutations that suppress the photosynthetic deficiency (spd mutations) of strains lacking cytochrome c2 (cyt c2) cause accumulation of a periplasmic cyt c2 isoform that has been designated isocytochrome c2 (isocyt c2). In this study, a new method for purification of both cyt c2 and isocyt c2 is described that uses periplasmic fluid as a starting material. In addition, antiserum to isocyt c2 has been used to demonstrate that all suppressor mutants contain an isocyt c2 of approximately 15 kDa. Western blot analysis indicates that isocyt c2 was present at lower levels in both wild-type and cyt c2 mutants than in spd-containing mutants. Although isocyt c2 is detectable under all growth conditions in wild-type cells, the highest level of isocyt c2 is present under aerobic conditions. Our results demonstrate that spd mutations increase the steady state level of isocyt c2 under photosynthetic conditions. Although the physiological function of isocyt c2 in wild-type cells is not known, we show that a nitrate-regulated protein in Rhodobacter sphaeroides f. sp. denitrificans also reacts with the isocyt c2 antiserum.
The cytochrome b-c1 complex from Rhodobacter sphaeroides was resolved into four protein subunits by a phenyl-Sepharose CL-4B column eluted with different detergents. Individual subunits were purified to homogeneity. Antibodies against subunit IV (Mr = 15,000) were raised and purified. These antibodies had a high titer with isolated subunit IV and with the b-c1 complex from R. sphaeroides. They inhibited 95% of the ubiquinol-cytochrome c reductase activity of the cytochrome b-c1 complex, indicating that subunit IV is essential for the catalytic function of this complex. When detergent-solubilized chromatopores were passed through an anti-subunit IV coupled Affi-Gel 10 column, no no ubiquinol-cytochrome c reductase activity was detected in the effluent, and four proteins, corresponding to the four subunits in the isolated complex, were adsorbed to the column. This indicated that subunit IV in an integral part of the cytochrome b-c1 complex. No change in the apparent Kms for Q2H2 and for cytochrome c was observed with anti-subunit IV treated complex. Antibodies against subunit IV had little effect on the stability of the ubisemiquinone radical in this complex, suggesting that they do not bind to the subunit near its ubiquinone-binding site.
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This chapter discusses genetic techniques used when working with photosynthetic bacteria, focusing on Rhodospirillaceae. Many photosynthetic bacteria are gram-negative and amenable, in varying degrees, to techniques for genetic analysis used in Escherichia coli and Salmonella typhimurium. Rhodospirillaceae are within the α and β subdivisions of the purple bacteria. They are either obligately or facultatively photosynthetic; the latter have a wide spectrum of growth modes. Rhodobacter species and Rhodospirillum rubrum grow well under both aerobic and anaerobic conditions. These organisms are capable of virtually all known biological energy transformations under anaerobic conditions. These organisms are capable of virtually all known biological energy transformations under anaerobic conditions. Hence, Rhodospirillaceae offer an important tool for genetic engineering.
Converging physiological, genetic, and biochemical studies have established the salient features of preprotein translocation across the plasma membrane of Escherichia coli. Translocation is catalyzed by two proteins, a soluble chaperone and a membrane-bound translocase. SecB, the major chaperone for export, forms a complex with preproteins. Complex formation inhibits side-reactions such as aggregation and misfolding and aids preprotein binding to the membrane surface. Translocase consists of functionally linked peripheral and integral membrane protein domains. SecA protein, the peripheral membrane domain of translocase, is the primary receptor for the SecB/preprotein complex. SecA hydrolyzes ATP, promoting cycles of translocation, preprotein release, Δµ~H+-dependent translocation, and rebinding of the preprotein. The membrane-embedded domain of translocase is the SecY/E protein. It has, as subunits, the SeeY and SecE polypeptides. The SecY/E protein stabilizes and activates SecA and participates in binding it to the membrane. SecA recognizes the leader domain of preproteins, whereas both SecA and SecB recognize the mature domain. Many proteins translocate without requiring SecB, and some proteins do not need translocase to assemble into the plasma membrane. Translocation is usually followed by endoproteolytic cleavage by leader peptidase. The availability of virtually every pure protein and cloned gene involved in the translocation process makes E. coli the premier organism for the study of translocation mechanisms.
The genes coding for the photosynthetic reaction center cytochrome c subunit (pufC) and the soluble cytochrome c2 (cycA) from the purple non-sulfur bacterium Rhodopseudomonas viridis were expressed in Escherichia coli. Biosynthesis of the reaction center cytochrome without a signal peptide resulted in the formation of inclusion bodies in the cytoplasm amounting to 14% of the total cellular protein. A series of plasmids coding for the cytochrome subunit with varying N-terminal signal peptides was constructed in attempts to achieve translocation across the E. coli cytoplasmic membrane and heme attachment. However, the two major recombinant proteins with N-termini corresponding to the signal peptide and the cytochrome were synthesized in E. coli as non-specific aggregates without heme incorporation. An increased ratio of precursor as compared to 'processed' apo-cytochrome was obtained when expression was carried out in a proteinase-deficient strain. Cytochrome c2 from R. viridis was synthesized in E. coli as a precursor associated with the cytoplasmic membrane. An expression plasmid was designed encoding the N-terminal part of the 33 kDa precursor protein of the oxygen-evolving complex of Photosystem II from spinach followed by cytochrome c2. Two recombinant proteins without heme were found to aggregate as inclusion bodies with N-termini corresponding to the signal peptide and the mature 33 kDa protein.
Plasmid pWS2 is an R68.45 chimera originally isolated as an R-prime which complemented the Rhodobacter sphaeroides bch-420 allele. Our experiments have shown that pWS2 is also able to complement a wide range of R. sphaeroides pigment and photosynthetic mutants employing nitrosoquanidine, transposon or insertion-generated mutations effecting puhA, puc, puf, cycA, bch, and crt genes. A combination of orthogonal-field-alternation gel electrophoresis, transverse alternating field gel electrophoresis, and conventional electrophoresis have been used to estimate the size of pWS2 at congruent to 168.3 +/- 3.5 kb. A restriction map of the congruent to 109 kb of R. sphaeroides insert DNA was generated by partial and complete restriction endonuclease digestion coupled with Southern hybridization analysis using either gene-specific or junction fragment probes. Genes encoding bacteriochlorophyll (Bchl)-binding proteins (pufBALMX, pucBA, and puhA), cytochrome c2 (cycA), and enzymes involved in Bchl (bch) and carotenoid (crt) biosynthesis have been shown to reside within a contiguous 53-kb region of the R. sphaeroides DNA present on pWS2. The puf operon lies at one end of the 53-kb segment, while the genes puhA, cycA, and pucBA, the latter two of which are located within congruent to 12.0 kb of each other, define the other end of this 53-kb region. The genetic and physical mapping data provided in this paper are discussed in terms of the similarities and differences in the organization of the photosynthetic gene cluster between R. sphaeroides and other photosynthetic bacteria as well as highlighting the use of pWS2 in studies of photosynthetic gene structure and function.
The structure of the photosynthetic reaction center (RC) from Rhodobacter sphaeroides was determined at 3.1-A resolution by the molecular replacement method, using the Rhodopseudomonas viridis RC as the search structure. Atomic coordinates were refined with the difference Fourier method and restrained least-squares refinement techniques to a current R factor of 22%. The tertiary structure of the RC complex is stabilized by hydrophobic interactions between the L and M chains, by interactions of the pigments with each other and with the L and M chains, by residues from the L and M chains that coordinate to the Fe2+, by salt bridges that are formed between the L and M chains and the H chain, and possibly by electrostatic forces between the ends of helices. The conserved residues at the N-termini of the L and M chains were identified as recognition sites for the H chain.
A highly active, large-scale preparation of ubiquinol:cytochrome c2 oxidoreductase (EC 1.10.2.2; cytochrome bc1 complex) has been obtained from Rhodobacter sphaeroides. The enzyme was solubilized from chromatophores by using dodecyl maltoside in the presence of glycerol and was purified by anion-exchange and gel filtration chromatography. The procedure yields 35 mg of pure bc1 complex from 4.5 g of membrane protein, and its consistently results in an enzyme preparation that catalyzes the reduction of horse heart cytochrome c with a turnover of 250-350 (mumol of cyt c reduced).(mumol of cyt c1)-1.s-1. The turnover number is at least double that of the best preparation reported in the literature [Ljungdahl, P. O., Pennoyer, J. D., Robertson, D. C., & Trumpower, B. L. (1987) Biochim. Biophys. Acta 891, 227-241]. The scale is increased 25-fold, and the yield is markedly improved by using this protocol. Four polypeptide subunits were observed by SDS-PAGE, with Mr values of 40K, 34K, 24K, and 14K. N-Terminal amino acid sequences were obtained for cytochrome c1, the iron-sulfur protein subunit, and for cytochrome b and were identical with the expected protein sequences deduced from the DNA sequence of the fbc operon, with the exceptions that a 22-residue fragment is processed off of the N-terminus of cytochrome c1 and the N-terminal methionine residue is cleaved off both the b cytochrome and iron-sulfur protein subunits. Western blotting experiments indicate that subunit IV is not a contaminating light-harvesting complex polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)
The cytochrome bc1 complex is the most widely occurring electron transfer complex capable of energy transduction. Cytochrome bc1 complexes are found in the plasma membranes of phylogenetically diverse photosynthetic and respiring bacteria, and in the inner mitochondrial membrane of all eucaryotic cells. In all of these species the bc1 complex transfers electrons from a low-potential quinol to a higher-potential c-type cytochrome and links this electron transfer to proton translocation. Most bacteria also possess alternative pathways of quinol oxidation capable of circumventing the bc1 complex, but these pathways generally lack the energy-transducing, protontranslocating activity of the bc1 complex. All cytochrome bc1 complexes contain three electron transfer proteins which contain four redox prosthetic groups. These are cytochrome b, which contains two b heme groups that differ in their optical and thermodynamic properties; cytochrome c1, which contains a covalently bound c-type heme; and a 2Fe-2S iron-sulfur protein. The mechanism which links proton translocation to electron transfer through these proteins is the proton motive Q cycle, and this mechanism appears to be universal to all bc1 complexes. Experimentation is currently focused on understanding selected structure-function relationships prerequisite for these redox proteins to participate in the Q-cycle mechanism. The cytochrome bc1 complexes of mitochondria differ from those of bacteria, in that the former contain six to eight supernumerary polypeptides, in addition to the three redox proteins common to bacteria and mitochondria. These extra polypeptides are encoded in the nucleus and do not contain redox prosthetic groups. The functions of the supernumerary polypeptides of the mitochondrial bc1 complexes are generally not known and are being actively explored by genetically manipulating these proteins in Saccharomyces cerevisiae.