New insights into the structure and dynamics of the TOM complex in mitochondria.

To date, there is no general physical model of the mechanism by which unfolded polypeptide chains with different properties are imported into the mitochondria. At the molecular level, it is still unclear how transit polypeptides approach, are captured by the protein translocation machinery in the outer mitochondrial membrane, and how they subsequently cross the entropic barrier of a protein translocation pore to enter the intermembrane space. This deficiency has been due to the lack of detailed structural and dynamic information about the membrane pores. In this review, we focus on the recently determined sub-nanometer cryo-EM structures and our current knowledge of the dynamics of the mitochondrial two-pore outer membrane protein translocation machinery (TOM core complex), which provide a starting point for addressing the above questions. Of particular interest are recent discoveries showing that the TOM core complex can act as a mechanosensor, where the pores close as a result of interaction with membrane-proximal structures. We highlight unusual and new correlations between the structural elements of the TOM complexes and their dynamic behavior in the membrane environment.

The monofunctional cobalamin biosynthesis enzyme precorrin-3B synthase (CobZRR) is essential for anaerobic photosynthesis in Rhodospirillum rubrum but not for aerobic dark metabolism.

The in vivo physiological role of the gene cobZ, which encodes precorrin-3B synthase, which catalyzes the initial porphyrin ring contraction step of cobalamin biosynthesis via the cob pathway, has been demonstrated here for the first time. Cobalamin is known to be essential for an early step of bacteriochlorophyll biosynthesis in anoxygenic purple bacteria. The cobZ (cobZRR) gene of the purple bacterium Rhodospirillum rubrum was localized to a 23.5 kb insert of chromosomal DNA contained on the cosmid pSC4. pSC4 complemented several mutants of bacteriochlorophyll and carotenoid biosynthesis, due to the presence of the bchCX and crtCDEF genes at one end of the cosmid insert, flanking cobZRR. A second gene, citB/tcuB, immediately downstream of cobZRR, shows homologies to both a tricarballylate oxidoreductase (tcuB) and a gene (citB) involved in signal transduction during citrate uptake. CobZRR shows extensive homology to the N-terminal domain of the bifunctional CobZ from Rhodobacter capsulatus, and the R. rubrum citB/tcuB gene is homologous to the CobZ C-terminal domain. A mutant, SERGK25, containing a terminatorless kanamycin interposon inserted into cobZRR, could not grow by anaerobic photosynthesis, but grew normally under dark, aerobic and microaerophilic conditions with succinate and fructose as carbon sources. The anaerobic in vivo activity of CobZ indicates that it does not require oxygen as a substrate. The mutant excreted large amounts of protoporphyrin IX-monomethylester, a brown precursor of bacteriochlorophyll biosynthesis. The mutant was complemented either by the cobZRR gene in trans, or when exogenous cobalamin was added to the medium. A deletion mutant of tcuB/citB did not exhibit the cob phenotype. Thus, a role for tcuB/citB in cobalamin biosynthesis could not be confirmed.

Random mutagenesis and overexpression of rhodopin-3,4-desaturase allows the production of highly conjugated carotenoids in Rhodospirillum rubrum.

The crtD gene of the purple bacterium Rhodospirillum rubrum, encoding rhodopin desaturase, was cloned into a broad-host range expression plasmid (pRKCAG53) and transferred to the R. rubrum crtD(-) mutant, ST4, which restored the wild-type phenotype and produced the carotenoid spirilloxanthin. pRKCAG53 was randomly mutated in an Escherichia coli mutator strain and then transferred to ST4 for selection of non-wild-type phenotypes. Strains containing the mutated expression plasmid exhibited two coloured phenotypes: a "brown" phenotype, corresponding to 3,4,3',4'-tetrahydrospirilloxanthin, arising from plasmids containing an inactivated crtD gene, and secondly, a "dark pink" phenotype. Absorption and mass spectra and HPLC analysis obtained from hexane extracts of brown mutants, confirmed the carotenoid assignment above. DNA sequence analysis of the crtD genes from the brown transconjugants showed frameshifts at the extreme C-terminus, suggesting that this domain forms part of the active site. Spectral analysis of the dark pink strains showed an additional, non-natural double bond formed at the carotenoid end, yielding the asymmetric carotenoids, 3,4,3',4'-tetradehydrorhodopin - and 3',4'-didehydroanhydrorhodovibrin, each containing 14 conjugated double bonds. For only two dark pink strains, was a mutation in crtD detected, in both cases at the N-terminus of CrtD. Otherwise, the higher conjugation was ascribed to an elevated plasmid copy number.

Assessing the performance of the four question abbreviated mental test in the acute geriatric setting.

BACKGROUND: NCognitive impairment is common amongst acute geriatric hospital admissions but detection is often poor and this is associated with worse outcomes. The four-question abbreviated mental test (AMT4) has previously been promoted nationally in the acute setting as a succinct assessment tool. However, a recent national dementia Commissioning for Quality and Innovation (CQUIN) goal recommends a single screening question followed by the tenquestion abbreviated mental test (AMT10). We aimed to evaluate the negative predictive value of the AMT4 within the acute setting by comparing it to three other validated tools. METHOD: We identified 100 acute medical admissions (>60 years old) with a negative AMT4 and administered the AMT10, six-item cognitive impairment test (6CIT) and confusion assessment method (CAM) within 24 hours of admission. RESULTS: Forty-six percent of the participants scored positively on at least one of the additional tests despite a negative AMT4. Forty-four patients had a positive 6CIT, 23 had a positive AMT10 and six had a positive CAM. Using the 6CIT as a diagnostic standard, tests of short-term memory had the greatest sensitivity and specificity for cognitive impairment. CONCLUSION: Nearly half of the participants had signs of cognitive impairment despite a negative AMT4. Consequently, there is a risk of under diagnosis with potentially serious consequences for morbidity and mortality. Tests of shortterm memory were strongly associated with cognitive impairment. We propose the addition of such a test in order to increase the sensitivity of the AMT4 without compromising its brevity and utility in the acute setting.

Computational prediction of extracellular loops of the Por39 outer membrane porin of Rhodospirillum rubrum suitable for epitope surface display.

Outer membrane porins from Gram-negative bacteria are established vehicles for the production of vaccines. Typically, one or more of the extracellular loops of a porin are replaced by a peptide encoding a foreign epitope, and recombinant porin is then used as a vaccine. However, many host strains are potentially pathogenic, and also produce toxic lipopolysaccharide (LPS), both of which are undesirable for safety reasons. In contrast, the outer membrane porins from photosynthetic, purple bacteria have no known human pathology and produce only weakly toxic LPS. The purple bacterium Rhodospirillum rubrum is well-suited for large-scale biotechnology, and expresses a major porin, Por39, which is a candidate for a vaccine platform. Unfortunately, the atomic structure of Por39 could not be determined so far, and Por39 shows only a weak homology to other porins of known structure, making the assignment of external loops difficult. Here, we construct a knowledge-based model of Por39 using secondary structure constraints from both the low sequence homology to the 2POR porin from Rhodobacter capsulatus, for which the X-ray structure is known, as well as those obtained using secondary structure prediction packages. The secondary structure predictions were used to constrain a three-dimensional model created using the I-TASSER package. The modelling procedure was validated by predicting the structure of 2POR using the same strategy, but excluding the 2POR X-ray structure from the I-TASSER database. The final Por39 model allows three external loops to be defined precisely, and could also be used to obtain an initial model for the closely related Por41 using molecular modelling. These structures provide a good starting point for the insertion of epitopes with vaccine potential.

High-level production of the industrial product lycopene by the photosynthetic bacterium Rhodospirillum rubrum.

The biosynthesis of the major carotenoid spirilloxanthin by the purple nonsulfur bacterium Rhodospirillum rubrum is thought to occur via a linear pathway proceeding through phytoene and, later, lycopene as intermediates. This assumption is based solely on early chemical evidence (B. H. Davies, Biochem. J. 116:93-99, 1970). In most purple bacteria, the desaturation of phytoene, catalyzed by the enzyme phytoene desaturase (CrtI), leads to neurosporene, involving only three dehydrogenation steps and not four as in the case of lycopene. We show here that the chromosomal insertion of a kanamycin resistance cassette into the crtC-crtD region of the partial carotenoid gene cluster, whose gene products are responsible for the downstream processing of lycopene, leads to the accumulation of the latter as the major carotenoid. We provide spectroscopic and biochemical evidence that in vivo, lycopene is incorporated into the light-harvesting complex 1 as efficiently as the methoxylated carotenoids spirilloxanthin (in the wild type) and 3,4,3',4'-tetrahydrospirilloxanthin (in a crtD mutant), both under semiaerobic, chemoheterotrophic, and photosynthetic, anaerobic conditions. Quantitative growth experiments conducted in dark, semiaerobic conditions, using a growth medium for high cell density and high intracellular membrane levels, which are suitable for the conventional industrial production in the absence of light, yielded lycopene at up to 2 mg/g (dry weight) of cells or up to 15 mg/liter of culture. These values are comparable to those of many previously described Escherichia coli strains engineered for lycopene production. This study provides the first genetic proof that the R. rubrum CrtI produces lycopene exclusively as an end product.

New Approach for the Construction and Calibration of Gas-Tight Setups for Biohydrogen Production at the Small Laboratory Scale.

Biohydrogen production in small laboratory scale culture vessels is often difficult to perform and quantitate. One problem is that commonly used silicon tubing and improvised plastic connections used for constructing apparatus are cheap and easy to connect but are generally not robust for gases such as hydrogen. In addition, this type of apparatus presents significant safety concerns. Here, we demonstrate the construction of hydrogen-tight apparatus using a commercially available modular system, where plastic tubing and connections are made of explosion-proof dissipative plastic material. Using this system, we introduce a gas chromatograph calibration procedure, which can be easily performed without necessarily resorting to expensive commercial gas standards for the calibration of hydrogen gas concentrations. In this procedure, the amount of hydrogen produced by the reaction of sodium borohydride with water in a closed air-filled bottle is deduced from the observed decrease of the oxygen partial pressure, using the ideal gas law. Finally, the determined calibration coefficients and the gas-tight apparatus are used for the analysis of simultaneous oxygen consumption and hydrogen production of the purple photosynthetic bacterium, Rhodospirillum rubrum, during semi-aerobic growth in the dark.

Excitation energy pathways in the photosynthetic units of reaction center LM- and H-subunit deletion mutants of Rhodospirillum rubrum.

Light-induced reaction dynamics of isolated photosynthetic membranes obtained from wild-type (WT) and reaction center (RC)-subunit deletion strains SPUHK1 (an H-subunit deletion mutant) and SK Delta LM (an (L+M) deletion mutant) of the purple non-sulphur bacterium Rhodospirillum rubrum have been investigated by femtosecond transient absorption spectroscopy. Upon excitation of the spirilloxanthin (Spx) S(2) state at 546 nm, of the bacteriochlorophyll Soret band at 388 nm and probing spectral regions, which are characteristic for carotenoids, similar dynamics in the SPUHK1, SK Delta LM and WT strains could be observed. The excitation of Spx S(2) is followed by the simultaneous population of the lower singlet excited states S(1) and S* which decay with lifetimes of 1.4 and 5 ps, respectively for the mutants, and 1.4 and 4 ps, respectively, for the wild-type. The excitation of the BChl Soret band is followed by relaxation into BChl lower excited states which compete with excitation energy transfer BChl-to-Spx. The deexcitation pathway BChl(Soret) --> Spx(S(2)) --> Spx(S(1)) occurs with the same transition rate for all investigated samples (WT, SPUHK1 and SK Delta LM). The kinetic traces measured for the Spx S(1) --> S(N) transition display similar behaviour for all samples showing a positive signal which increases within the first 400 fs (i.e. the time needed for the excitation energy to reach the Spx S(1) excited state) and decays with a lifetime of about 1.5 ps. This suggests that the Spx excited state dynamics in the investigated complexes do not differ significantly. Moreover, a longer excited state lifetime of BChl for SPUHK1 in comparison to WT was observed, consistent with a photochemical quenching channel present in the presence of RC. For long delay times, photobleaching of the RC special pair and an electrochromic blue shift of the monomeric BChl a can be observed only for the WT but not for the mutants. The close similarity of the excited state decay processes of all strains indicates that the pigment geometry of the LH1 complex in native membranes is unaffected by the presence of an RC and allows us to draw a model representation of the WT, SK Delta LM and SPUHK1 PSU complexes.

Modeling the electron transport chain of purple non-sulfur bacteria.

Purple non-sulfur bacteria (Rhodospirillaceae) have been extensively employed for studying principles of photosynthetic and respiratory electron transport phosphorylation and for investigating the regulation of gene expression in response to redox signals. Here, we use mathematical modeling to evaluate the steady-state behavior of the electron transport chain (ETC) in these bacteria under different environmental conditions. Elementary-modes analysis of a stoichiometric ETC model reveals nine operational modes. Most of them represent well-known functional states, however, two modes constitute reverse electron flow under respiratory conditions, which has been barely considered so far. We further present and analyze a kinetic model of the ETC in which rate laws of electron transfer steps are based on redox potential differences. Our model reproduces well-known phenomena of respiratory and photosynthetic operation of the ETC and also provides non-intuitive predictions. As one key result, model simulations demonstrate a stronger reduction of ubiquinone when switching from high-light to low-light conditions. This result is parameter insensitive and supports the hypothesis that the redox state of ubiquinone is a suitable signal for controlling photosynthetic gene expression.

The Methoxylated, Highly Conjugated C40 Carotenoids, Spirilloxanthin and Anhydrorhodovibrin, Can Be Separated Using High Performance Liquid Chromatography with Safe and Environmentally Friendly Solvents.

High performance liquid chromatography (HPLC) is a frequently used technique in carotenoid research. So far, however, little attention has been paid to the fact that many of the organic solvents used in HPLC separation of highly apolar C40 carotenoids impose a significant threat to both health (especially for women) and the general laboratory environment. Here, we developed a solvent combination capable of allowing high-resolution HPLC separation of the C40 carotenoid, spirilloxanthin, and all of its biosynthetic precursors beginning with phytoene, using relatively safe, environmentally friendly solvents. We show that separation of spirilloxanthin and its precursors anhydrorhodovibrin and lycopene using modern ultra-high performance chromatography (UHPLC) poses particular problems for apolar carotenoid separation, due to the long residence times in the sample delivery system, which facilitates carotenoid aggregation. We resolved these problems by developing the solvent delivery combination acetone/acetonitrile/isopropanol/methanol (65/30/5/2 (v/v/v/v)), which allows excellent column separation using the safe isocratic solvent system methanol/tetrahydrofuran (98/2 (v/v)). We also demonstrate that the development strategy for optimizing a solvent system for carotenoid separation can be well-described by the use of the average dielectric constant of the total sample delivery solvent, and present a formal method for analysis of the efficiency of separation.

Redox-state dynamics of ubiquinone-10 imply cooperative regulation of photosynthetic membrane expression in Rhodospirillum rubrum.

It is now well established that, for photosynthetic bacteria, the aerobic-to-microaerophilic transition activates the membrane-bound sensor kinase RegB, which subsequently phosphorylates the transcriptional activator RegA, thereby inducing elevated levels of intracellular photosynthetic membranes. The mechanism of RegB activation--in particular, the role of ubiquinone-10--is controversial at present. One problem here is that very limited quantitative in vivo data for the response of the ubiquinone redox state to different cultivation conditions exist. Here, we utilize Rhodospirillum rubrum to study the correlation of the quinone redox state to the expression level of photosynthetic membranes and determine an effective response function directly. Our results show that changes in the photosynthetic membrane levels between 50 and 95% of that maximally attainable are associated with only a twofold change in the ubiquinol/ubiquinone ratio and are not necessarily proportional to the total levels of either quinone or [NAD(+) + NADH]. There is no correlation between the redox potentials of the quinone and pyridine nucleotide pools. Hill function analysis of the photosynthetic membrane induction in response to the quinone redox state suggests that the induction process is highly cooperative. Our results are probably generally applicable to quinone redox regulation in bacteria.

Initial binding process of the membrane insertase YidC with its substrate Pf3 coat protein is reversible.

The binding of the inner membrane insertase YidC from Escherichia coli to its substrate, the Pf3 coat protein, was examined in vitro by fluorescence spectroscopy. Purified YidC protein was solubilized with the lipid-like detergent n-dodecylphosphocholine and noncovalently labeled with 1-anilino-naphthalene-8-sulfonate (ANS), whereas the Pf3 coat protein was kept in solution by the addition of 10% (v/v) isopropanol to the buffer. The binding of Pf3 coat protein was analyzed by fluorescence quenching of ANS bound to YidC. All binding curves showed a strict hyperbolic form at pH values between 9.0 and 5.0, indicating a reversible and noncooperative binding between YidC and its substrate. Analysis of the data revealed a dissociation constant K D for the binding process in the range of 1 microM. The pH profile of the K D values suggests that the binding of the Pf3 coat protein is dominated by hydrophobic interactions. The titration experiments provide strong evidence for a conformational change of the insertase upon binding a Pf3 coat protein molecule.

A Modified Kulka Micromethod for the Rapid and Safe Analysis of Fructose and 1-Deoxy-d-xylulose-5-phosphate.

The Kulka resorcinol assay (Kulka, R.G., Biochemistry 1956, 63, 542⁻548) for ketoses has been widely used in the literature but suffers from two major disadvantages: (a) it employs large amounts of potentially harmful reagents for a general biology laboratory environment; and (b) in its original formulation, it is unsuited for modern high-throughput applications. Here, we have developed a modified Kulka assay, which contains a safer formulation, employing approx. 5.4 M HCl in 250 µL aliquots, and is suitable for use in high-throughput systems biology or enzymatic applications. The modified assay has been tested extensively for the measurement of two ketoses-fructose (a common substrate in cell growth experiments) and 1-deoxy-d-xylulose-5-phosphate (DXP), the product of the DXP-synthase reaction-which until now has only been assayable using time-consuming chromatographic methods or radioactivity. The Kulka microassay has a sensitivity of 0⁻250 nmol fructose or 0⁻500 nmol DXP. The assay is suitable for monitoring the consumption of fructose in bacterial growth experiments but is too insensitive to be used directly for the measurement of DXP in in vitro enzyme assays. However, we show that after concentration of the DXP-enzyme mix by butanol extraction, the Kulka resorcinol method can be used for enzyme assays.

A rapid procedure for the in situ assay of periplasmic, PQQ-dependent methanol dehydrogenase in intact single bacterial colonies.

Mechanistic details of methanol oxidation catalyzed by the periplasmically-located pyrroloquinoline quinone-dependent methanol dehydrogenase of methylotrophs can be elucidated using site-directed mutants. Here, we present an in situ colony assay of methanol dehydrogenase, which allows robotic screening of large populations of intact small colonies, and regrowth of colonies for subsequent analysis.

Quantum Redirection of Antenna Absorption to Photosynthetic Reaction Centers.

The early steps of photosynthesis involve the photoexcitation of reaction centers (RCs) and light-harvesting (LH) units. Here, we show that the historically overlooked excitonic delocalization across RC and LH pigments results in a redistribution of absorption amplitudes that benefits the absorption cross section of the optical bands associated with the RC of several species. While we prove that this redistribution is robust to the microscopic details of the dephasing between these units in the purple bacterium Rhodospirillum rubrum, we are able to show that the redistribution witnesses a more fragile, but persistent, coherent population dynamics which directs excitations from the LH toward the RC units under incoherent illumination and physiological conditions. Even though the redirection does not seem to affect importantly the overall efficiency in photosynthesis, stochastic optimization allows us to delineate clear guidelines and develop simple analytic expressions in order to amplify the coherent redirection in artificial nanostructures.

N-(omega-(4-(2-methoxyphenyl)piperazin-1-yl)alkyl)carboxamides as dopamine D2 and D3 receptor ligands.

The dopamine D(3) receptor is recognized as a potential therapeutic target for the treatment of various neurological and psychiatric disorders. Targetting high affinity and D(3) versus D(2) receptor-preferring ligands, the partial agonist BP 897 was taken as a lead structure. Variations in the spacer and the aryl moiety led to N-alkylated 1-(2-methyoxyphenyl)piperazines with markedly improved affinity and selectivity. Molecular modeling studies supported the structural development. Pharmacophore models for dopamine D(2) and D(3) receptor ligands were developed from their potentially bioactive conformation and were compared in order to get insight into molecular properties of importance for D(2)/D(3) receptor selectivity. For the 72 compounds presented here, an extended and more linear conformation in the aliphatic or aryl spacers turned out to be crucial for dopamine D(3) receptor selectivity. Structural diversity in the aryl moiety (benzamides, heteroarylamides, arylimides) had a major influence on (sub)nanomolar D(3) receptor affinity, which was optimized with more rigid aryl acrylamide derivatives. Compound 38 (ST 280, (E)-4-iodo-N-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)cinnamoylamide) displayed a most promising pharmacological profile (K(i) (hD(3)) = 0.5 nM; K(i) (hD(2L)) = 76.4 nM; selectivity ratio of 153), and above that, compound 38 offered the prospect of a novel radioligand as a pharmacological tool for various D(3) receptor-related in vitro and in vivo investigation.

A rapid method for the extraction and analysis of carotenoids and other hydrophobic substances suitable for systems biology studies with photosynthetic bacteria.

A simple, rapid, and inexpensive extraction method for carotenoids and other non-polar compounds present in phototrophic bacteria has been developed. The method, which has been extensively tested on the phototrophic purple non-sulphur bacterium Rhodospirillum rubrum, is suitable for extracting large numbers of samples, which is common in systems biology studies, and yields material suitable for subsequent analysis using HPLC and mass spectroscopy. The procedure is particularly suitable for carotenoids and other terpenoids, including quinones, bacteriochlorophyll a and bacteriopheophytin a, and is also useful for the analysis of polar phospholipids. The extraction procedure requires only a single step extraction with a hexane/methanol/water mixture, followed by HPLC using a Spherisorb C18 column, with a mobile phase consisting of acetone-water and a non-linear gradient of 50%-100% acetone. The method was employed for examining the carotenoid composition observed during microaerophilic growth of R. rubrum strains, and was able to determine 18 carotenoids, 4 isoprenoid-quinones, bacteriochlorophyll a and bacteriopheophytin a as well as four different phosphatidylglycerol species of different acyl chain compositions. The analytical procedure was used to examine the dynamics of carotenoid biosynthesis in the major and minor pathways operating simultaneously in a carotenoid biosynthesis mutant of R. rubrum.

The reaction center H subunit is not required for high levels of light-harvesting complex 1 in Rhodospirillum rubrum mutants.

The gene (puhA) encoding the H subunit of the reaction center (RC) was deleted by site-directed interposon mutagenesis by using a kanamycin resistance cassette lacking transcriptional terminators to eliminate polar effects in both the wild-type strain Rhodospirillum rubrum S1 and the carotenoid-less strain R. rubrum G9. The puhA interposon mutants were incapable of photoheterotrophic growth but grew normally under aerobic chemoheterotrophic conditions. Absorption spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the RCs were absent. In minimal medium and also in modified medium containing succinate and fructose, the light-harvesting 1 complex (LH1) levels of the S1-derived mutants were about 70 to 100% of the wild-type levels in the same media. The correct assembly of LH1 in the membrane and the pigment-pigment interaction were confirmed by near-infrared circular dichroism spectroscopy. LH1 formation was almost absent when the carotenoid-less G9-derived puhA mutants were grown in standard minimal medium, suggesting that carotenoids may stabilize LH1. In the fructose-containing medium, however, the LH1 levels of the G9 mutants were 70 to 100% of the parental strain levels. Electron micrographs of thin sections of R. rubrum revealed photosynthetic membranes in all mutants grown in succinate-fructose medium. These studies indicate that the H subunit of the RC is necessary neither for maximal formation of LH1 nor for photosynthetic membrane formation but is essential for functional RC assembly.

Microaerophilic cooperation of reductive and oxidative pathways allows maximal photosynthetic membrane biosynthesis in Rhodospirillum rubrum.

The purple nonsulfur bacterium Rhodospirillum rubrum has been employed to study physiological adaptation to limiting oxygen tensions (microaerophilic conditions). R. rubrum produces maximal levels of photosynthetic membranes when grown with both succinate and fructose as carbon sources under microaerophilic conditions in comparison to the level (only about 20% of the maximum) seen in the absence of fructose. Employing a unique partial O(2) pressure (pO(2)) control strategy to reliably adjust the oxygen tension to values below 0.5%, we have used bioreactor cultures to investigate the metabolic rationale for this effect. A metabolic profile of the central carbon metabolism of these cultures was obtained by determination of key enzyme activities under microaerophilic as well as aerobic and anaerobic phototrophic conditions. Under aerobic conditions succinate and fructose were consumed simultaneously, whereas oxygen-limiting conditions provoked the preferential breakdown of fructose. Fructose was utilized via the Embden-Meyerhof-Parnas pathway. High levels of pyrophosphate-dependent phosphofructokinase activity were found to be specific for oxygen-limited cultures. No glucose-6-phosphate dehydrogenase activity was detected under any conditions. We demonstrate that NADPH is supplied mainly by the pyridine-nucleotide transhydrogenase under oxygen-limiting conditions. The tricarboxylic acid cycle enzymes are present at significant levels during microaerophilic growth, albeit at lower levels than those seen under fully aerobic growth conditions. Levels of the reductive tricarboxylic acid cycle marker enzyme fumarate reductase were also high under microaerophilic conditions. We propose a model by which the primary "switching" of oxidative and reductive metabolism is performed at the level of the tricarboxylic acid cycle and suggest how this might affect redox signaling and gene expression in R. rubrum.

Circular symmetry of the light-harvesting 1 complex from Rhodospirillum rubrum is not perturbed by interaction with the reaction center.

The effect of the interaction of the reaction center (RC) upon the geometrical arrangement of the bacteriochlorophyll a (BChla) pigments in the light-harvesting 1 complex (LH1) from Rhodospirillum rubrum has been examined using single molecule spectroscopy. Fluorescence excitation spectra at 1.8 K obtained from single detergent-solubilized as well as single membrane-reconstituted LH1-RC complexes showed predominantly (>70%) a single broad absorption maximum at 880-900 nm corresponding to the Q(y) transition of the LH1 complex. This absorption band was independent of the polarization direction of the excitation light. The remaining complexes showed two mutually orthogonal absorption bands in the same wavelength region with moderate splittings in the range of DeltaE = 30-85 cm(-1). Our observations are in agreement with simulated spectra of an array of 32 strongly coupled BChla dipoles arranged in perfect circular symmetry possessing only a diagonal disorder of