Rhodospirillum rubrum possesses a variant of the bchP gene, encoding geranylgeranyl-bacteriopheophytin reductase

The bchP gene product of Rhodobacter sphaeroides is responsible for the reduction of the isoprenoid moiety of bacteriochlorophyll (Bchl) from geranylgeraniol (GG) to phytol; here, we show that this enzyme also catalyzes the reduction of the isoprenoid moiety of bacteriopheophytin (Bphe). In contrast, we demonstrate that a newly identified homolog of this gene in Rhodospirillum rubrum encodes an enzyme, GG-Bphe reductase, capable of reducing the isoprenoid moiety of Bphe only. We propose that Rhodospirillum rubrum is a naturally occurring bchP mutant and that an insertion mutation may have been the initial cause of a partial loss of function. Normal BchP function can be restored to Rhodospirillum rubrum, creating a new transconjugant strain possessing Bchl esterified with phytol. We speculate on the requirement of Rhodospirillum rubrum for phytylated Bphe and on a potential link between the absence of LH2 and of phytylated Bchl from the wild-type bacterium. The identification of a second role for the fully functional BchP in catalyzing the synthesis of phytylated Bphe strongly suggests that homologs of this enzyme may be similarly responsible for the synthesis of phytylated pheophytin in organisms possessing photosystem 2. In addition to bchP, other members of a photosynthesis gene cluster were identified in Rhodospirillum rubrum, including a bchG gene, demonstrated to encode a functional Bchl synthetase by complementation of a Rhodobacter sphaeroides mutant.     

Light and oxygen regulation of the synthesis of bacteriochlorophylls a and c in Chloroflexus aurantiacus.

Control of the synthesis of bacteriochlorophylls (Bchls) a and c by light and oxygen was studied in Chloroflexus aurantiacus grown in batch or chemostat culture with serine as the growth-limiting substrate. For comparison, inhibition by gabaculine of the formation of selected tetrapyrroles was studied. The inhibitory effect of gabaculine decreased in the following order of tetrapyrrole formation: coproporphyrin greater than Bchl c greater than Bchl a. Not only did addition of 5-aminolevulinate (ALA) reverse the inhibition by gabaculine, it also caused an increase in Bchl c content when the cultures grew at high concentrations of ALA. Inhibition of Bchl a, Bchl c, and coproporphyrin formation by oxygen was similar to inhibition by gabaculine. Addition of ALA to aerated cultures led to significant accumulation of coproporphyrin. These results suggest that oxygen inhibits tetrapyrrole formation at a site before ALA formation. Control by light was studied with chemostat cultures transferred from 5 klx to 25 klx. This resulted in only a transient increase of the protein level of the culture, while specific contents of Bchls c and a and the ratio Bchl c/Bchl a decreased to lower steady states. However, the specific content of coproporphyrin increased. Addition of ALA to chemostat cultures adapted to 50 klx increased specific coproporphyrin and Bchl c contents by factors of about 20 and 4, respectively, while the specific Bchl a content was only slightly increased and protein levels were unaffected. Increasing the serine concentration caused an initial increase in the specific Bchl c content, which returned to the original value as soon as the protein content had attained its maximal level. These results suggest that light does not control ALA formation as strictly as oxygen and that competition of biomass formation and tetrapyrrole synthesis for common precursors may be influenced by light.     

Solvation effect of bacteriochlorophyll excitons in light-harvesting complex LH2

We have characterized the influence of the protein environment on the spectral properties of the bacteriochlorophyll (Bchl) molecules of the peripheral light-harvesting (or LH2) complex from Rhodobacter sphaeroides. The spectral density functions of the pigments responsible for the 800 and 850 nm electronic transitions were determined from the temperature dependence of the Bchl absorption spectra in different environments (detergent micelles and native membranes). The spectral density function is virtually independent of the hydrophobic support that the protein experiences. The reorganization energy for the B850 Bchls is 220 cm(-1), which is almost twice that of the B800 Bchls, and its Huang-Rhys factor reaches 8.4. Around the transition point temperature, and at higher temperatures, both the static spectral inhomogeneity and the resonance interactions become temperature-dependent. The inhomogeneous distribution function of the transitions exhibits less temperature dependence when LH2 is embedded in membranes, suggesting that the lipid phase protects the protein. However, the temperature dependence of the fluorescence spectra of LH2 cannot be fitted using the same parameters determined from the analysis of the absorption spectra. Correct fitting requires the lowest exciton states to be additionally shifted to the red, suggesting the reorganization of the exciton spectrum.     

A reaction center-light-harvesting 1 complex (RC-LH1) from a Rhodospirillum rubrum mutant with altered esterifying pigments: characterization by optical spectroscopy and cryo-electron microscopy

Introduction of the bchP gene from Rhodobacter sphaeroides encoding geranylgeranyl reductase into Rhodospirillum rubrum alters the esterification of the bacteriochlorophylls so that phytol is used instead of geranylgeraniol. The resulting transconjugant strain of Rs. rubrum grows photosynthetically, showing that phytolated Bchla can substitute for the native pigment in both the reaction center (RC) and the light-harvesting 1 (LH1) complexes. This genetic manipulation perturbs the native carotenoid biosynthetic pathway; several biosynthetic intermediates are assembled into the core complex and are capable of energy transfer to the bacteriochlorophylls. RC-LH1 complexes containing phytolated Bchla were analyzed by low temperature absorption and fluorescence spectroscopy and circular dichroism. These show that phytolated Bchls can assemble in vivo into the photosynthetic apparatus of Rs. rubrum and that the newly introduced phytol tail provokes small perturbations to the Bchls within their binding sites in the LH1 complex. The RC-LH1 core complex was purified from membranes and reconstituted into well ordered two-dimensional crystals with a p4212 space group. A projection map calculated to 9 A shows clearly that the LH1 ring from the mutant is composed of 16 subunits that surround the reaction center and that the diameter of this complex is in close agreement with that of the wild-type LH1 complex.     

Role of the C-terminal extrinsic region of the alpha polypeptide of the light-harvesting 2 complex of Rhodobacter sphaeroides: a domain swap study

The LH1 and LH2 complexes of Rhodobacter sphaeroides form ring structures of 16 and 9 protomers, respectively, comprising alpha and beta polypeptides, bacteriochlorophylls (Bchl), and carotenoids. Using the LH2 complex as a starting point, two chimeric LH complexes were constructed incorporating the alphaC-terminal domain of either the Rb. sphaeroides LH1 complex or the Rhodospirillum molischianum LH2 complex. The LH1 domain swap produced a new red-shifted component that comprised approximately 30% of the total absorbance. In the LH1alpha C-terminal mutant this new red-shifted species acts as the terminal emitter, with the new emission maximum located 10 nm further to the red than for the WT. Raman spectroscopy indicates that a fraction of the B850 Bchls is involved in relatively weak H-bonds, possibly involving the alphaTrp(+11) residue within the new alphaC-terminus, consistent with a more LH1-like character for one of the Bchls. The CD data indicate that the domain swaps have perturbed the native arrangement of the B850 Bchls, including the site energy difference between the alpha- and beta-bound Bchls. Thus, the normal energetic structure of the ring system has been disrupted, with one component blue shifted due to the presumed loss of an H-bond donor and the other red shifted by the influence of the new alphaC-terminal domain. The dichotomous response of the mutants to the carotenoids incorporated, spheroidenone or neurosporene, strongly suggests that the C-terminal region of the alpha polypeptide is involved in binding a carotenoid. The projection map of the LH1alpha C-terminal mutant complex was determined in negative stain at 25 A resolution, and it shows a diameter of 53 A, compared to 50 A for the WT. Hence these new spectral properties have not been accompanied by an alteration in ring size.     

Polypeptides and bacteriochlorophyll organization in the light-harvesting complex B850 of Rhodobacter sphaeroides R-26.1

The light-harvesting complex (LHC) B850 from Rhodobacter sphaeroides was dissociated into several fragments by treatment with sodium dodecyl sulfate. The molecular weight of each fragment was determined by using transverse polyacrylamide gel electrophoresis under nondenaturing conditions and gel filtration techniques. Four B850 LHCs were observed, having molecular weights of 60,000, 72,000-75,000, 105,000, and 125,000-145,000, and two small bacteriochlorophyll (Bchl)-polypeptide complexes having molecular weights of 6000-8000 and 12,000-14,000. Each of the B850 complexes contains ca. one Bchl a for each 6.5-kDa protein. The optical absorption and circular dichroism of the B850 LHCs recorded directly from the gels are similar to those measured previously for a 22-24-kDa B850 LHCs by Sauer and Austin [(1978) Biochemistry 17, 2011-2019]. These data, combined with studies of other groups, indicate that the smallest LHC in LH1 and LH2 is a Bchl-polypeptide tetramer. Each tetramer contains two Bchl dimers that probably have the structure of P-860, the primary electron donor in Rhodobacter sphaeroides, and two alpha-beta-polypeptide pairs. Interactions among the paired Bchls shift their individual Qy transitions from 780-800 to 850-860 nm, and interactions among two such pairs induce the circular dichroism signal of the LHCs. Three Bchl-polypeptide tetramers probably form a dodecamer having C3 symmetry, and six such dodecamers organize into a large hexagon that can accommodate one or two reaction center complexes.     

Isolation, characterization, and comparison of a ubiquitous pigment-protein complex consisting of a reaction center and light-harvesting bacteriochlorophyll proteins present in purple photosynthetic bacteria

Protein complexes (photochemical reaction complex; PR complex) bound to both light-harvesting bacteriochlorophyll-1 (LH-Bchl-1) and reaction center Bchl (RC-Bchl) were purified from Rhodospirillum rubrum (wild and carotenoid-less), Rhodopseudomonas sphaeroides (wild), and Chromatium vinosum (wild). Another protein complex (LH-2 complex) bound to LH-Bchl-2 was also purified from Rps. sphaeroides. The bacteria were grown in the presence of a [14C]amino acid mixture. The purification procedure included molecular-sieve chromatography in the presence of cholate-deoxycholate, and non-equilibrated isoelectric electrophoresis with 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate. The purified complexes were separated into their constituent proteins by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The molar ratios of the proteins were determined by comparing their radioactivities divided by their molecular weights after consideration of the molecular masses of the complexes. The PR complexes all contained per mol: 1 mol each of RC H-, M-, and L-subunits, 10-13 (probably 12) mol each of two other proteins with molecular weights of 11-12K and 8-11K, 28-32 mol Bchl, 13-15 mol carotenoids (except in the carotenoid-less mutant), 2.6-3.9 mol ubiquinone (or menaquinone in Chr. vinosum), and 53-79 mol phosphate without phospholipid. The LH-2 complex contained per mol: 1 mol 52K protein, about 13 (probably 12) mol each of 9K and 8K proteins, 30 mol Bchl, 10 mol carotenoids, and 38 mol phosphate without phospholipid. The PR complexes and LH-2 complex showed similar X-ray diffraction patterns, implying that they had similar, highly organized molecular structures.     

Growth rate and control of development of the photosynthetic apparatus in Chloroflexus aurantiacus

Chloroflexus aurantiacus was grown photoheterotrophically in a chemostat in order to study the influence of growth rate on the formation of bacteriochlorophyll a (Bchl a) which represents the membrane-bound photosynthetic pigment complexes, and of Bchl c which represents the light harvesting pigment-proteins of the chlorosome. Steady state cell protein levels as well as specific Bchl a contents increased linearly and specific Bchl c contents exponentially when the dilution rate, representing growth rate, was decreased. In spite of differences in the light intensities, continuous cultures growing at comparable growth rates and densities exhibited comparable specific contents of both Bchls and largely identical molar ratios of Bchl c/Bchl a. The growth rate of constantly illuminated batch cultures was varied by changing the concentration of growth-limiting nutrients. Cultures growing at higher growth rates showed higher cell densities but lower specific Bchl levels as well as lower molar ratios of Bchl c/Bchl a than cultures growing at low growth rate. Determination of the light energy flux required for half-maximal saturation of photosynthetic activity (light dependent proton extrusion) by chemostat cultures showed a dependency of that activity by the content of cellular Bchl c. In summary, the results suggest that, growth rate or a factor regulating growth rate, rather than light affected specific Bchl levels and because of the increasing molar ratio of Bchl c to Bchl a, the light harvesting capacity and photosynthetic efficiency of the photosynthetic apparatus.     

Structure and X-ray amino acid sequence of a bacteriochlorophyll a protein from Prosthecochloris aestuarii refined at 1.9 A resolution

The structure of the water-soluble bacteriochlorophyll a protein (Bchl protein) from the green photosynthetic bacterium Prosthecochloris aestuarii has been refined at 1.9 A resolution to a crystallographic residual of 18.9%. The refinement was carried out without knowledge of the amino acid sequence and has led to an "X-ray sequence". The structure consists of seven Bchl molecules enclosed within a protein "bag" and the refinement supports the general conformation of the molecule described previously. The refinement also supports the previous suggestion that the ligands to the seven Bchl magnesiums are, respectively, five histidines, a carbonyl oxygen from the polypeptide backbone of the protein, and a bound water molecule. The conformations of the seven Bchl head-groups are described in detail. In two cases the magnesium atoms are approximately 0.48 A "below" the plane of the conjugated macrocycle while in the other five cases the atoms are, on average, 0.48 A "above" the plane. The acetyl ring substituents are more-or-less coplanar with the dihydrophorbin macrocycle, consistent with a previous resonance Raman study. The conjugated atoms in each of the seven macrocycles have significant departures from strict planarity. These deviations are similar for Bchls 1, 2 and 3 (class I) and are also similar for Bchls 4, 5, 6 and 7 (class II). Ethylchlorophillide also belongs to class II. The out-of-plane deformations for the class I and class II bacteriochlorophylls appear to correspond to two distinct modes of bending or curvature of the dihydrophorbin macrocycle.     

Regulation of tetrapyrrole synthesis by light in chemostat cultures of Rhodobacter sphaeroides.

Control of bacteriochlorophyll (Bchl), magnesium protoporphyrin monomethyl ester (MgPME), cytochromes, and coproporphyrin by light was studied with chemostat cultures of Rhodobacter sphaeroides growing at a constant dilution rate. By increasing the growth-limiting light energy flux from 10 to 55 W/m2, specific Bchl contents decreased from 19.3 to 7.9 nmol/mg of protein. This was strictly proportional to a decrease in the ratio of B800-850 to B875 light-harvesting complexes. MgPME levels increased from 1.5 to 5.3 nmol/mg of protein, while cytochrome as well as coproporphyrin levels stayed constant at 0.46 and 1.95 nmol/mg of protein, respectively. Since in chemostat cultures steady-state levels of a product represent the rate of synthesis, these results infer only slight control of the rate-limiting step of total tetrapyrrol formation by light. In substrate-limited cultures MgPME was accumulated when growth and Bchl formation approached substrate saturation. This suggests that light controls a second step, i.e., MgPME conversion, whenever too much precursor is available, owing to the low sensitivity of the initial step of control. MgPME was preferentially localized in a subcellular fraction with high contents of B875 complexes. A second fraction exhibiting increased contents of B800-850 complexes lacked significant levels of MgPME. These results are discussed in terms of localization of Bchl synthesis in the membrane system of R. sphaeroides.     

Influence of carotenoid molecules on the structure of the bacteriochlorophyll binding site in peripheral light-harvesting proteins from Rhodobacter sphaeroides

In the LH2 proteins from Rhodobacter (Rb.) sphaeroides, the hydrogen bonds between the bacteriochlorophyll (Bchl) molecules and their proteic binding sites exhibit a strong variance with respect to carotenoid content and type. In the absence of the carotenoid molecule, such as in the LH2 from Rb. sphaeroides R26.1, the void in the protein structure induces a significant reorganization of the binding site of both Bchl molecules responsible for the 850 nm absorption, which is not observed when the 800 nm absorbing Bchl is selectively removed from these complexes. FT Raman spectra of LH2 complexes from Rb. sphaeroides show that the strength of the hydrogen bond between the 850 nm absorbing Bchl bound to the alpha polypeptide and the tyrosine alpha(45) depends precisely on the chemical nature of the bound carotenoid. These results suggest that the variable extremity of the carotenoid is embedded in these LH2 complexes, lying close to the interacting Bchl molecules. In the LH2 from Rhodopseudomonas acidophila, the equivalent part of the rhodopin glucoside, which bears the glucose group, lies close to the amino terminal of the antenna polypeptide. This contrast suggests that the structure of the carotenoid binding site in LH2 complexes strongly depends on the bacterial species and/or on the chemical nature of the bound carotenoid.     

Dichroism of bacteriochlorophyll in chromatophores of photosynthetic bacteria.

The dichroism was measured in films of air-dried and, consequently, flattened chromatophores of Chromatium vinosum, Rhodopseudomonas sphaeroides and Rhodospirillum rubrum. The values (deltaA/A) of dichroism in C. vinosum were found to be -1.05 at 590 nm and 0.75 in the near infrared region. The values of dichroism in R. sphaeroides were -0.70 at 590 nm and 0.80 at 870 nm. The values of dichroism in R. rubrum were -1.45 at 590 nm and 0.97 at 870 nm.     

Energy-transfer dynamics in three light-harvesting mutants of Rhodobacter sphaeroides: a picosecond spectroscopy study

Picosecond absorption spectroscopy has been used to investigate energy-transfer dynamics within the LH1 and LH2 light-harvesting complexes of three mutants of Rhodobacter sphaeroides. We demonstrate that both complexes are inhomogeneous; each contains a specialized pigment pool which absorbs maximally at a longer wavelength. Within LH2 (mutant NF57), Bchl850 transfers energy to Bchl870 in 39 +/- 9 ps; within LH1 (mutants M21 and M2192), energy is transferred from Bchl875 to Bchl896 in 22 +/- 4 and 35 +/- 5 ps, respectively. Examination of the decay of induced absorption anisotropy indicates that each of these specialized pools exists in a state which is highly organized with respect to the remainder of the pigments. Such an arrangement may facilitate and direct energy migration toward the reaction center.     

Physical mapping and functional assignment of the geranylgeranyl-bacteriochlorophyll reductase gene, bchP, of Rhodobacter sphaeroides

The bacteriochlorophyll of the purple photosynthetic bacterium Rhodobacter sphaeroides is esterified with phytol. The presence of this alcohol moiety is essential for the correct assembly of the photosynthetic apparatus. Despite this, and the fact that R. sphaeroides is widely used for the study of structure-function relationships in photosynthesis, the molecular genetics of the steps in which the alcohol is added and modified have not previously been investigated in this organism. Sequencing near the center of the photosynthesis gene cluster has now revealed the existence of an open reading frame encoding a putative 394-amino-acid polypeptide displaying strong homology with the products of a number of genes from other photosynthetic organisms, each proposed to be responsible for the reduction of the alcohol moiety of (bacterio)chlorophyll to phytol. An R. sphaeroides transposon mutant in this gene, bchP, possessed a structurally modified photosystem assembled with bacteriochlorophyll esterified with geranylgeraniol, rather than with phytol, implying that the product of this gene was geranylgeranyl-bacteriochlorophyll reductase. This identification was confirmed by the performance of in vitro assays using heterologously expressed protein, providing the first direct demonstration of the activity of a bchP gene product.     

Genetic and physical mapping of the Rhodobacter sphaeroides photosynthetic gene cluster from R-prime pWS2

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.     

Single-Molecule Spectroscopy Reveals that Individual Low-Light LH2 Complexes from Rhodopseudomonas palustris 2.1.6. Have a Heterogeneous Polypeptide Composition

Rhodopseudomonas palustris belongs to the group of purple bacteria that have the ability to produce LH2 complexes with unusual absorption spectra when they are grown at low-light intensity. This ability is often related to the presence of multiple genes encoding the antenna apoproteins. Here we report, for the first time to our knowledge, direct evidence that individual low-light LH2 complexes have a heterogeneous αβ-apoprotein composition that modulates the site energies of Bchl a molecules, producing absorption bands at 800, 820, and 850 nm. The arrangement of the Bchl a molecules in the “tightly coupled ring” can be modeled by nine αβ-Bchls dimers, such that the Bchls bound to six αβ-pairs have B820-like site energies and the remaining Bchl a molecules have B850-like site energies. Furthermore, the experimental data can only be satisfactorily modeled when these six αβ-pairs with B820 Bchl a molecules are distributed such that the symmetry of the assembly is reduced to C₃. It is also clear from the measured single-molecule spectra that the energies of the electronically excited states in the mixed B820/850 ring are mainly influenced by diagonal disorder.     

On the influence of pigment-protein interactions on the energy transfer processes in photosynthetic membrane structures. 2. LH2 complex of Rhodobacter sphaeroides

Low-temperature heterogeneous absorption and circular dichroism spectra of the Rb. sphaeroides LH2 complexes are calculated within the framework of the mini-exciton theory and diagonal static random disorder for the pure electronic transitions of the monomeric Bchl molecules. The coupling of Bchl molecules with the surrounding amino acid residues has been shown to change both the exciton distribution between the pigment molecules in each of the exciton states. The value of the delocalization index depends on the excitation wavelength and varies between 2-6 Bchl molecules. The optical transitions occurring at 780-790 and 820 nm have been found to be strongly mixed so that all Bchl molecules of the LH2 complex predetermine absorption in these spectral regions. On the other hand, absorption at 800 and 850 nm is mainly determined by the cycles of 9 and 18 Bchl molecules, respectively. Thus, the light energy absorbed by the B800 molecules at 800 nm is transferred to the B850 molecules by the interlevel exciton relaxation processes due to the population of the heavily mixed 820-nm exciton levels. The width of the heterogeneous absorption band for the cyclic monomeric aggregate has been shown to decrease as compared with the monomeric absorption band by square root(Ndel) time, where Ndel is the mean number of pigments over which the exciton is delocalized within the excited absorption band.     

Distinctive properties of bacilliform photosynthetic heliobacteria

Abstract Heliobacterium chlorum and Heliobacillus mobilis are closely related N₂-fixing anoxyphoto-trophs that contain bacteriochlorophyll g (Bchl g ) as the major photopigment. In the presence of O₂ and light, the absorbance peak in the infra-red (788 nm) of this novel photoreceptor disappears and absorbance at 670 nm increases simultaneously. These optical changes appear to be due to a photoisomerization reaction which converts Bchl g to a form of green-plant chlorophyll a (in which farnesol replaces phytol). In addition to this unusual property, the Gram-negative heliobacteria present biochemical features (16S RNA base sequence and peptidoglycan structure) indicating an evolutionary relationship with some Gram-positive bacteria. In comparison to H. chlorum, H. mobilis grows more robustly and shows a much lower tendency to spheroplast and lyse; accordingly, H. mobilis is better suited for further investigations on the biology and biochemistry of these exceptional prokaryotes.     

Fermentation of pyruvate by 7 species of phototrophic purple bacteria]

The dark, anaerobic fermentation of pyruvate under growth conditions was examined with the following species of phototrophic purple bacteria: Rhodospirillum rubrum strains Ha and S1, Rhodopseudomonas gelatinosa strain 2150, Rhodopseudomonas acidophila strain 7050, Rhodopseudomonas palustris strain ATCC 17001, Rhodopseudomonas capsulata strains Kb1 and 6950, Rhodopseudomonas sphaeroides strain ATCC 17023, and Chromatium vinosum strain D. Fermentation balances were established for all experiments. Under fermentative conditions cell protein and dry weight increased only slightly, if at all. The species differed considerably in their fermentative activity; R. rubrum and R. gelatinosa exhibited the highest rates (2-8 mumoles pyruvate/mg protein-h). R. acidophila and R. capsulata showed an intermediate fermentation rate (0.4--2.0 mumoles pyruvate/mg protein-h), while the other strains tested fermented at quite low rates (0.2-0.4 mumoles pyruvate/mg protein-h). The extremes of fermentation times were from 30-380 hours. Based on the products of fermentation which were formed in addition to acetate, formate, and CO2, the species can be grouped as follows: a) R. rubrum, R. gelatinosa, and R. sphaeroides additionally form propionate. b) R. gelatinosa, R. palustris, R. capsulata, R. sphaeroides, and C. vinosum additionally form lactate. R. palustris also produces butyrate. c) R. acidophila and R. capsulata additionally form much 2,3-butanediol, acetoin, and diacetyl. Small amounts of acetoin were formed by the rest of the strains. A comparison of the fermentation of pyruvate by normal and starved cells (4 days in the light without a carbon source) of R. rubrum and R. gelatinosa shows that the latter ferment more slowly and produce less acetate and formate, but more propionate or lactate. The fermentation of pyruvate by R. rubrum was also studied in cultures in which the pH fell (7.2--6.6). Compared with the fermentation at neutral pH (7.3, 7.4), the following differences were found: a slower fermentation rate, an increased production of dry weight, an increased formation of propionate, but a reduced formation of acetate and a very low production of formate.     

The pet operon,encoding the prosthetic group-containing subunits of the cytochrome bc1 complex,of the purple sulfur bacterium Chromatium vinosum

The pet operon, encoding the prosthetic group-containing subunits of the cytochrome bc ₁ complex of the purple sulfur bacterium Chromatium vinosum, has been cloned and sequenced. The 5 to 3 order of the C. vinosum genes is: petA, encoding the Rieske iron-sulfur protein; petB, encoding cytochrome b; and petC, encoding cytochrome c₁. Cytochrome b is the best conserved subunit of the C. vinosum complex, when compared to the corresponding proteins from four photosynthetic purple non-sulfur bacteria (70 to 74% identity). Identities for the C. vinosum Rieske protein and those from purple non-sulfur bacteria range from 60 to 64%. The C-terminal region of the C. vinosum Rieske protein is quite similar to those of purple non-sulfur bacteria, while the N-terminal region is more closely related to mitochondrial Rieske proteins of organisms such as Neurospora crassa. Cytochrome c₁ is the least well-conserved protein of the C. vinosum cytochrome bc₁ complex, with identities ranging from 49 to 51% when compared to the corresponding proteins from purple non-sulfur bacteria. A well-conserved negatively-charged region of the cytochromes c₁ of the purple non-sulfur bacteria, thought to be involved in binding the electron acceptor for the complex, cytochrome c₂, is absent in C. vinosum cytochrome c₁. A positive Southern hybridization using a probe constructed from the Rhodobacter sphaeroides fbcQ gene, which codes for a fourth subunit of the cytochrome bc₁ complex in that bacterium, suggests the presence of a homologous gene in C. vinosum.