appA, a novel gene encoding a trans-acting factor involved in the regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1.

A new gene, the product of which is involved in the regulation of photosynthesis gene expression in the anoxygenic photosynthetic bacterium Rhodobacter sphaeroides 2.4.1, has been identified. The isolation of this gene, designated appA (activation of photopigment and puc expression), was based on its ability, when provided in extra copies, to partially suppress mutations in the two-component PrrB-PrrA regulatory system. The presence of extra copies of the appA gene in either prrB, prrA, or wild-type strains resulted in an activation of puc::lacZ expression under aerobic conditions. Constructed AppA null mutants did not grow photosynthetically and were impaired in the synthesis of both bacteriochlorophyll and carotenoids, as well as the structural proteins of the photosynthetic spectral complexes. When grown anaerobically in the dark, these mutants accumulated bacteriochlorophyll precursors. The expression of lacZ fusions to several photosynthesis genes and operons, including puc, puf, and bchF, was decreased in the AppA mutant strains in comparison with the wild type. To examine the role of AppA involvement in bacteriochlorophyll biosynthesis, we inactivated an early gene, bchE, of the bacteriochlorophyll pathway in both wild-type and AppA- mutant backgrounds. The double mutant, AppA- BchE-, was found to be severely impaired in photosynthesis gene expression, similar to the AppA- BchE+ mutant and in contrast to the AppA+ BchE- mutant. This result indicated that AppA is more likely involved in the regulation of expression of the bch genes than in the biosynthetic pathway per se. The appA gene was sequenced and appears to encode a protein of 450 amino acids with no obvious homology to known proteins.     

Bacteriochlorophyll-dependent expression of genes for pigment-binding proteins in<Emphasis Type="Italic"> Rhodobacter capsulatus</Emphasis> involves the RegB/RegA two-component system

Expression of the puf and puc operons, which encode proteins of the photosynthetic apparatus of Rhodobacter capsulatus, is regulated by oxygen. A drop in the oxygen tension in the environment leads to an increase in the levels of puf and puc mRNAs. In strains lacking bacteriochlorophyll (Bchl) due to mutations in bch genes, the rise in puf and puc mRNA levels observed on reduction of oxygen tension is much less pronounced than in wild-type cells, indicating co-regulation of the syntheses of pigments and pigment-binding proteins. Here we show that Bchl synthesis also affects the expression of the bchC gene, which codes for a subunit of bacteriochlorophyll synthase, suggesting an autoregulatory mechanism for the Bchl biosynthetic pathway. Furthermore, our data provide evidence that the RegB/RegA two-component system, which is known to play a central role in oxygen-controlled expression of photosynthesis genes, is also involved in the Bchl-dependent regulation. Mutant strains which do not synthesize RegB or RegA show similar oxygen-dependent puf and puc expression in the presence and absence of Bchl. Our results support the view that the RegB/RegA system can directly or indirectly sense whether Bchl synthesis takes place or not.     

Semiaerobic induction of bacteriochlorophyll synthesis in the green bacterium Chloroflexus aurantiacus.

Comparison of Chloroflexus aurantiacus J-10-fl cells by freeze-fracture electron microscopy showed that cell shape and dimensions did not depend on oxygen tension or light intensity during growth. The major morphological difference between cells cultured anaerobically in the light and aerobically in the dark was the absence of chlorosomes in aerobically grown cells. C. aurantiacus cells cultured aerobically in the dark began bacteriochlorophyll synthesis immediately when shifted to either phototrophic or semiaerobic conditions. Cells adapting to phototrophic conditions grew to the same density and synthesized as much bacteriochlorophyll as nonadapting phototrophic cultures grown at the same light intensity. Cells adapting to reduced oxygen tension (semiaerobic conditions) in the dark entered an 8- to 12-h growth lag during which the bacteriochlorophyll content increased significantly. Despite variations in the initial bacteriochlorophyll content and in the length of the growth lag, the amounts of bacteriochlorophyll a and c were constant at the end of the semiaerobic growth lag. At later times during adaptation to semiaerobic conditions, after growth resumed, variations in the ratio of bacteriochlorophyll c/bacteriochlorophyll a were observed and suggested independent regulation of the two bacteriochlorophylls.     

The formation of bacteriochlorophyll · protein complexes of the photosynthetic apparatus of Rhodopseudomonas capsulata during early stages of development

Cells of Rhodopseudomonas capsulata cultivated at an oxygen partial pressure of 400 mmHg in the dark contained 0.1 nmol or less total bacteriochlorophyll per mg membrane protein. The bacteriochlorophyll was found in the reaction center (10 pmol bacteriochlorophyll/mg membrane protein) and in the light harvesting bacteriochlorophyll I but not in the light harvesting bacteriochlorophyll II. Formation of the photosynthetic apparatus in those cells was induced by incubation at a very low oxygen tension in the dark. Reaction center bacteriochlorophyll and light harvesting bacteriochlorophyll increased three fold after 60 min of incubation at 1–2 mmHg (pO₂). Light harvesting bacteriochlorophyll II increased strongly after 60 min and became dominating after 90 min of incubation. The total bacteriochlorophyll content doubled every 30 min, but synthesis of reaction center bacteriochlorophyll proceeded at much lower rates. Consequently the size of the photosynthetic unit (total bacteriochlorophyll/reaction center bacteriochlorophyll) increased from 15 to 52 during 150 min of incubation. The proteins of the photosynthetic apparatus were synthesized concomitantly with bacteriochlorophyll.Cells which were incubated at 0.5 mmHg (pO₂) do not grow but form the photosynthetic apparatus. During the first hours of incubation light harvesting bacteriochlorophyll I and reaction center bacteriochlorophyll were the dominant bacteriochlorophyll species, but light harvesting bacteriochlorophyll II was synthesized only in small amounts. Total bacteriochlorophyll and reaction center bacteriochlorophyll increased from 30 min up until 210 min of incubation more than 10 fold. The final concentrations of total bacteriochlorophyll and reaction center bacteriochlorophyll were 8.6 nmol and 0.26 nmol per mg membrane protein, respectively. The three protein components of the reaction centers (mol. wts. 28 000, 24 000 and 21 000) and the protein of the light harvesting I complex (mol. wt. 12 000) were incorporated simultaneously. The protein of band 1 (mol. wt. 14 000) which was present in the isolated light harvesting complex II, was synthesized only in very small amounts. The proteins of bands 3 and 4 (mol. wt. 10 000 and 8000) however, which were shown to be associated with light harvesting bacteriochlorophyll II, were synthesized in noticeable amounts as was light harvesting bacteriochlorophyll II. In addition a protein with an apparent molecular weight of 45 000 showed a strong incorporation of ¹⁴C-labeled amino acids. This protein comigrates with one protein which was found to be associated with a green pigment excreted during incubation at 0.5 Torr into the medium. The in vivo-absorption maxima of this pigment complex were 660, 590, 540, 417 and 400 nm. The succinate oxidase and the NADH oxidase seemed to be incorporated into the newly formed intracytoplasmic membrane only in very small amounts. Thus, reaction center and light harvesting bacteriochlorophyll and their associated proteins were simultaneously synthesized, whereas light harvesting complex II is the variable part of the photosynthetic apparatus.     

Oxygen does not directly regulate carotenoid biosynthesis in Rhodopseudomonas capsulata.

We examined the role of bacteriochlorophyll synthesis on the regulation of carotenoid synthesis in Rhodopseudomonas capsulata. Strains capable of making bacteriochlorophyll accumulated greater amounts of carotenoids under low oxygen than they did under high oxygen. However, strains unable to produce bacteriochlorophyll did not regulate their carotenoid production in response to changes in oxygen tension. This indicates that oxygen does not directly regulate carotenoid production.     

Control of the formation of bacteriochlorophyll,and B 875- and B 850-bacteriochlorophyll complexes in Rhodopseudomonas sphaeroides mutant strain H5

Rhodopseudomonas sphaeroides mutant H5 lacking 5-aminolevulinic acid synthase was employed to study the control of the formation of total bacteriochlorophyll as well as of the B875- and B850-bacteriochlorophyll protein complexes. The organisms were grown phototrophically in a chemostat where cell protein formation was limited by iron ions and bacteriochlorophyll by 5-aminolevulinic acid. 0.07 mol of bacteriochlorophyll was formed per mol of 5-amino-levulinic acid consumed. This stoichiometric relationship was not influenced by a twelve-fold variation in light energy flux. However, cell protein levels increased and, consequently, cellular specific bacteriochlorophyll contents decreased with increases in light energy flux. The ratio of B875- to B850-pigment protein complexes was inversely proportional to the velocity of 5-aminolevulinic acid supply (mol per cell protein and time) which in this system equals the velocity of 5-aminolevulinic acid consumption and the velocity of bacteriochlorophyll formation. Light had no direct effect on the ratio of B875- per B850-pigment complexes but an indirect effect via its control of protein formation. Changes in the ratio of the two pigment complexes resulted from the fact that significantly lower amounts of 5-aminolevulinic acid supplied per protein and time were required to saturate the system assembling the B875-complexes than that assembling the B850-complexes. The data suggest lack of light-dependent control in the formation of bacteriochlorophyll and its complexes subsequent to the 5-aminolevulinic acid pool.     

THE PHOTOSYNTHETIC APPARATUS OF RHODOSPIRILLUM MOLISCHIANUM

By varying the light intensity and temperature during growth it is possible to obtain cultures of Rhodospirillum molischianum in which the specific bacteriochlorophyll contents differ by as much as fivefold. We used such cultures to compare the changes in the electron microscopic appearance of the cells with the changes in the amount and bacteriochlorophyll content of chromatophore material isolated from cell extracts. The cells contained a variable number of internal membranes which are invaginations of the cell membrane. The shape, size, number, and arrangement of the infoldings varied as the specific bacteriochlorophyll content of the cells changed. In cells with little bacteriochlorophyll, the invaginations were mostly tubular. In cells with larger amounts of bacteriochlorophyll, the invaginations were disc-shaped and the discs were appressed together in stacks of 2 to 10 discs each. Variations in the number of discs per stack could be accounted for by a simple statistical model. The average area per disc increased with increasing bacteriochlorophyll content. Quantitative estimations of the relative volumes occupied by membranes in cells with four different bacteriochlorophyll contents showed that the amount of internal membrane alone had no direct relationship with the bacteriochlorophyll content of the cells; however, the total amount of membrane (cell membrane plus internal membrane) was directly proportional to the bacteriochlorophyll content. The specific bacteriochlorophyll content of isolated chromatophore material was proportional to the bacteriochlorophyll content of whole cells; the total amount of chromatophore material was independent of the bacteriochlorophyll content of whole cells. Several possible explanations of this paradoxical discrepancy between the electron microscope observations and the analytical results are discussed.     

THE PREPARATION AND PROPERTIES OF BACTERIAL CHROMATOPHORE FRACTIONS

Chromatophore material from the bacterium Rhodopseudomonas spheroides was freed of ribosomes by centrifugation in 27 per cent RbCl and then separated into "heavy" and "light" fractions by centrifugation through a sucrose gradient. The fractions differed from one another in the following ways. (a) The isopycnic density of the heavy fraction was between 1.15 and 1.18 gm/ml and that of the light fraction was 1.14 gm/ml. (b) The heavy fraction was able to bind ribosomes; the light fraction was not. (c) The light fraction was homogeneous in the ultracentrifuge and had a sedimentation constant, extrapolated to infinite dilution, of 153 s_20,w. The heavy fraction was grossly heterogeneous. (d) Both the amount of bacteriochlorophyll relative to protein and the ratio of bacteriochlorophyll to carotenoids were greater in the light fraction. (e) The spectra of the two fractions in the near infra-red were different. Comparisons of the chromatophore fractions from cells with different amounts of bacteriochlorophyll showed that the specific bacteriochlorophyll contents of the two fractions did not change to the same extent as did that of the whole cells. The amount of heavy fraction from pigmented cells was roughly independent of the cellular pigment content and was about equal to that from pigment-free cells. The amount of light fraction depended on the pigment content of the cells; no light fraction was obtained from cells devoid of bacteriochlorophyll. The cytochrome complements of both fractions underwent quantitative as well as qualitative changes with varying growth conditions. The size of the photosynthetic unit in R. spheroides appeared to increase as the total cellular bacteriochlorophyll content increased; however, the number of units per light fraction particle remained constant.     

prrA, a putative response regulator involved in oxygen regulation of photosynthesis gene expression in Rhodobacter sphaeroides.

A new locus, prrA, involved in the regulation of photosynthesis gene expression in response to oxygen, has been identified in Rhodobacter sphaeroides. Inactivation of prrA results in the absence of photosynthetic spectral complexes. The prrA gene product has strong homology to response regulators associated with signal transduction in other prokaryotes. When prrA is present in multiple copies, cells produce light-harvesting complexes under aerobic growth conditions, suggesting that prrA affects photosynthesis gene expression positively in response to oxygen deprivation. Analysis of the expression of puc::lacZ fusions in wild-type and PrrA- cells revealed a substantial decrease in LacZ expression in the absence of prrA under all conditions of growth, especially when cells were grown anaerobically in the dark in the presence of dimethyl sulfoxide. Northern (RNA) and slot blot hybridizations confirmed the beta-galactoside results for puc and revealed additional positive regulation of puf, puhA, and cycA by PrrA. The effect of truncated PrrA on photosynthesis gene expression in the presence of low oxygen levels can be explained by assuming that PrrA may be effective as a multimer. PrrA was found to act on the downstream regulatory sequences (J. K. Lee and S. Kaplan, J. Bacteriol. 174:1146-1157, 1992) of the puc operon regulatory region. Finally, two spontaneous prrA mutations that abolish prrA function by changing amino acids in the amino-terminal domain of the protein were isolated.     

Mutants of Rhodospirillum rubrum Obtained After Long-Term Anaerobic, Dark Growth

Rhodospirillum rubrum S(1) cells were grown for more than 100 generations under strict anaerobic, dark conditions in liquid medium with sodium pyruvate. During this time, growth became nonpigmented. When cells were streaked onto the surface of solid growth medium in anaerobic bottles and placed in the dark, a few light-red colonies developed, but the majority was nonpigmented. Mutants were obtained from colonies selected on the basis of pigmentation and bacteriochlorophyll a content. The growth, ultrastructure, and light reactivity of two mutants were examined. Mutant C synthesized bacteriochlorophyll a (7.2 mumoles per mg of protein), altered membrane structures, and chromatophores during dark growth. Examination of light-induced changes of the absorption spectrum of this mutant suggested that only an electron transport pathway, which included the low potential cytochrome-like pigment C428, could be detected. Mutant C grew anaerobically in the light, whereas mutant G1 was light sensitive and produced only trace amounts of bacteriochlorophyll a (0.6 mumole per ml of protein). Poorly pigmented G1 cells contained unusual membrane structures. When dark-grown G1 colonies were placed in the light, deep-red colored papillae developed in the nonpigmented colonies. During anaerobic, dark growth with sodium pyruvate, both C and G1 synthesized poly-beta-hydroxybutyrate and produced acetate, carbon dioxide, and hydrogen gas.     

Regulation of anaerobic carbon monoxide oxidation activity in Rhodocyclus gelatinosus

Rhodocyclus gelatinosus grows anaerobically at the expense of carbon monoxide (CO). The CO-oxidation system was substrate-induced and in CO/light, cells grew at an exponential rate with ever increasing amounts of CO:MV oxidoreductase activity (the measure of CO oxidation). Once strain 1 reached a high cell density, the concentration of CO became limiting and gas oxidation activity suddenly decreased. Cell growth continued unaffected. To help explain this, it appeared that strain 1 variably used both CO oxidation and photometabolism to support growth in CO/light. Light intensity determined the upper limit of amounts of CO:MV oxidoreductase in a culture, while intermediate amounts were regulated by CO concentration. Thus, in darkness, cells produced the maximum CO oxidation activity, whereas in growth-saturating light, the minimum limit occurred. The lower the levels of CO:MV oxidoreductase in cells, the greater the content of bacteriochlorophyll. In this manner, strain 1 grew with a generation time of 6.7 independent of light intensity.     

Isolation and characterization of light harvesting bacteriochlorophyll.protein complexes from Rhodopseudomonas capsulata

The isolation of two native light harvesting bacteriochlorophyl.protein complexes from Rhodopseudomonas capsulata is described. The light harvesting bacteriochlorophyll I (B 875) has been isolated from the blue-green mutant A1a+ lacking both carotenoids and light harvesting bacteriochlorophyll II. Light harvesting bacteriochlorophyll I is associated with a protein (light harvesting band 2) of 12 000 molecular weight. Light harvesting bacteriochlorophyll II complex has been isolated from the mutant Y5 lacking a reaction center and light harvesting bacteriochlorophyll I. Light harvesting bacteriochlorphyll II (B 800 + 850) together with carotenoids is associated with two polypeptides (light harvesting bands 3 and 4) having molecular weights of about 8000 and 10 000 (sodium dodecyl sulfate polyacrylamide gel electrophoresis). A third protein (light harvesting band 1) is in the purified light harvesting II fraction (mol. wt. approx. 14 000), but not associated with bacteriochlorophyll or carotenoids. The amino acid composition of the 3 antenna pigment II proteins is given. The polarity of these proteins was found to be 48%. From the amino acid composition the following molecular weights were calculated band 1: 17 350, band 3: 13 350 and band 4: 10 500.