Coupled reductive and oxidative sulfur cycling in the phototrophic plate of a meromictic lake.

Mahoney Lake represents an extreme meromictic model system and is a valuable site for examining the organisms and processes that sustain photic zone euxinia (PZE). A single population of purple sulfur bacteria (PSB) living in a dense phototrophic plate in the chemocline is responsible for most of the primary production in Mahoney Lake. Here, we present metagenomic data from this phototrophic plate--including the genome of the major PSB, as obtained from both a highly enriched culture and from the metagenomic data--as well as evidence for multiple other taxa that contribute to the oxidative sulfur cycle and to sulfate reduction. The planktonic PSB is a member of the Chromatiaceae, here renamed Thiohalocapsa sp. strain ML1. It produces the carotenoid okenone, yet its closest relatives are benthic PSB isolates, a finding that may complicate the use of okenone (okenane) as a biomarker for ancient PZE. Favorable thermodynamics for non-phototrophic sulfide oxidation and sulfate reduction reactions also occur in the plate, and a suite of organisms capable of oxidizing and reducing sulfur is apparent in the metagenome. Fluctuating supplies of both reduced carbon and reduced sulfur to the chemocline may partly account for the diversity of both autotrophic and heterotrophic species. Collectively, the data demonstrate the physiological potential for maintaining complex sulfur and carbon cycles in an anoxic water column, driven by the input of exogenous organic matter. This is consistent with suggestions that high levels of oxygenic primary production maintain episodes of PZE in Earth's history and that such communities should support a diversity of sulfur cycle reactions.

Environmental constraints defining the distribution, composition, and evolution of chlorophototrophs in thermal features of Yellowstone National Park.

Chlorophotoautotrophy, the use of chlorophylls to convert light energy into chemical energy for carbon dioxide fixation, is the primary metabolic process linking the inorganic and organic carbon pools on Earth. To understand the potential effects of various environmental constraints on the evolution of chlorophototrophy better, we studied the distribution, diversity, and abundance of chlorophylls and genes involved in their synthesis along geothermal gradients in Yellowstone National Park, Wyoming. Genes involved in chlorophyll biosynthesis were constrained to temperatures of less than ~70 °C and were only detected at this elevated temperature when the pH was in the circumneutral to alkaline range. The upper temperature limit for the detection of chlL/bchL(1) and bchY(2) decreased systematically with increasingly acidic pH, an observation likely attributable to sulfide, which upon oxidation, generates acidic spring water and reduces the availability of bicarbonate the preferred source of inorganic carbon for phototrophs. Spring pH was also the best predictor of the phylogenetic diversity of chlL/bchL communities. The phylogenetic similarity of chlL/bchL genes between sites was significantly correlated with that of chlorophylls. The predominance of chlorophyll a and bacteriochlorophyll a among extracted pigments was consistent with predominance of chlL/bchL genes affiliated with the Cyanobacteria and Chloroflexiales, respectively, and might be related to the fact that the majority of these organisms are photoautotrophs. Together, these results suggest that a combination of temperature, pH, and/or sulfide influences the distribution, diversity, and evolution of chlorophotrophs and the chlorophylls that they synthesize.

Biosynthesis of the biomarker okenone: χ-ring formation.

Purple sulfur bacteria (PSB) mainly occur in anoxic aquatic and benthic environments, where they play important roles in cycling carbon and sulfur. Many PSB characteristically produce the unique keto-carotenoid, okenone, which is important not only for its light absorption and photoprotection properties but also because of its diagenesis product, okenane, which is a biomarker for ancient sediments derived from anoxic environments. The specific methylation pattern of the χ-ring of okenane is unlikely to be formed by diagenetic processes and should therefore reflect an enzymatic activity from okenone biosynthesis. This study describes two enzymes that produce the χ-ring of okenone, the only structural element of okenone preserved in okenane. Genes encoding enzymes of carotenogenesis were identified in the draft genome sequence of an okenone-producing PSB, Thiodictyon sp. strain CAD16. Two divergently transcribed genes encoded a CrtY-type lycopene cyclase and a CrtU/CruE-type γ-carotene desaturase/methyltransferase. Expression of crtY in Escherichia coli showed that this gene encoded a lycopene cyclase that produced γ-carotene as the only product. Although the sequence of the γ-carotene desaturase/methyltransferase was more similar to CrtU sequences of green sulfur bacteria than to CruE sequences of cyanobacteria, expression of the crtU gene in Chlorobaculum tepidum showed that the enzyme produced carotenoids with χ-rings rather than φ-rings. Phylogenetic analysis of the carotene desaturase/methyltransferases revealed that enzymes capable of converting ß-rings to χ-rings have independently evolved at least two times. These results indicate that it probably will not be possible to deduce the activity of carotene desaturase/methyltransferases solely from sequence data.

X-ray scattering and electron cryomicroscopy study on the effect of carotenoid biosynthesis to the structure of Chlorobium tepidum chlorosomes.

Chlorosomes, the main antenna complexes of green photosynthetic bacteria, were isolated from null mutants of Chlorobium tepidum, each of which lacked one enzyme involved in the biosynthesis of carotenoids. The effects of the altered carotenoid composition on the structure of the chlorosomes were studied by means of x-ray scattering and electron cryomicroscopy. The chlorosomes from each mutant strain exhibited a lamellar arrangement of the bacteriochlorophyll c aggregates, which are the major constituents of the chlorosome interior. However, the carotenoid content and composition had a pronounced effect on chlorosome biogenesis and structure. The results indicate that carotenoids with a sufficiently long conjugated system are important for the biogenesis of the chlorosome baseplate. Defects in the baseplate structure affected the shape of the chlorosomes and were correlated with differences in the arrangement of lamellae and spacing between the lamellar planes of bacteriochlorophyll aggregates. In addition, comparisons among the various mutants enabled refinement of the assignments of the x-ray scattering peaks. While the main scattering peaks come from the lamellar structure of bacteriochlorophyll c aggregates, some minor peaks may originate from the paracrystalline arrangement of CsmA in the baseplate.

Backbone dynamics of plastocyanin in both oxidation states. Solution structure of the reduced form and comparison with the oxidized state.

A model-free analysis based on (15)N R(1), (15)N R(2), and (15)N-(1)H nuclear Overhauser effects was performed on reduced (diamagnetic) and oxidized (paramagnetic) forms of plastocyanin from Synechocystis sp. PCC6803. The protein backbone is rigid, displaying a small degree of mobility in the sub-nanosecond time scale. The loops surrounding the copper ion, involved in physiological electron transfer, feature a higher extent of flexibility in the longer time scale in both redox states, as measured from D(2)O exchange of amide protons and from NH-H(2)O saturation transfer experiments. In contrast to the situation for other electron transfer proteins, no significant difference in the dynamic properties is found between the two redox forms. A solution structure was also determined for the reduced plastocyanin and compared with the solution structure of the oxidized form in order to assess possible structural changes related to the copper ion redox state. Within the attained resolution, the structure of the reduced plastocyanin is indistinguishable from that of the oxidized form, even though small chemical shift differences are observed. The present characterization provides information on both the structural and dynamic behavior of blue copper proteins in solution that is useful to understand further the role(s) of protein dynamics in electron transfer processes.

Chromosomal gene inactivation in the green sulfur bacterium Chlorobium tepidum by natural transformation.

Conditions for inactivating chromosomal genes of Chlorobium tepidum by natural transformation and homologous recombination were established. As a model, mutants unable to perform nitrogen fixation were constructed by interrupting nifD with various antibiotic resistance markers. Growth of wild-type C. tepidum at 40 degrees C on agar plates could be completely inhibited by 100 microg of gentamicin ml(-1), 2 microg of erythromycin ml(-1), 30 microg of chloramphenicol ml(-1), or 1 microg of tetracycline ml(-1) or a combination of 300 microg of streptomycin ml(-1) and 150 microg of spectinomycin ml(-1). Transformation was performed by spotting cells and DNA on an agar plate for 10 to 20 h. Transformation frequencies on the order of 10(-7) were observed with gentamicin and erythromycin markers, and transformation frequencies on the order of 10(-3) were observed with a streptomycin-spectinomycin marker. The frequency of spontaneous mutants resistant to gentamicin, erythromycin, or spectinomycin-streptomycin was undetectable or significantly lower than the transformation frequency. Transformation with the gentamicin marker was observed when the transforming DNA contained 1 or 3 kb of total homologous flanking sequence but not when the transforming DNA contained only 0.3 kb of homologous sequence. Linearized plasmids transformed at least an order of magnitude better than circular plasmids. This work forms a foundation for the systematic targeted inactivation of genes in C. tepidum, whose 2.15-Mb genome has recently been completely sequenced.

Photosystem stoichiometry and state transitions in a mutant of the cyanobacterium Synechococcus sp. PCC 7002 lacking phycocyanin.

Phycobilisomes (PBS) function as light-harvesting antenna complexes in cyanobacteria, red algae and cyanelles. They are composed of two substructures: the core and peripheral rods. Interposon mutagenesis of the cpcBA genes of Synechococcus sp. PCC 7002 resulted in a strain (PR6008) lacking phycocyanin and thus the ability to form peripheral rods. Difference absorption spectroscopy of whole cells showed that intact PBS cores were assembled in vivo in the cpcBA mutant strain PR6008. Fluorescence induction measurements demonstrated that the PBS cores are able to deliver absorbed light energy to photosystem (PS) II, and fluorescence induction transients in the presence of DCMU showed that PR6008 cells could perform a state 2 to state 1 transition with similar kinetics to that of the wild-type cells. Thus, PBS core assembly, light-harvesting functions and energy transfer to PS I were not dependent upon the assembly of the peripheral rods. The ratio of PS II:PS I in the PR6008 cells was significantly increased, nearly twice that of the wild-type cells, possibly a result of long-term adaptation to compensate for the reduced antenna size of PS II. However, the ratio of PBS cores:chlorophyll remained unchanged. This result indicates that approximately half of the PS II reaction centers in the PR6008 cells had no closely associated PBS cores.

Electron transfer may occur in the chlorosome envelope: the CsmI and CsmJ proteins of chlorosomes are 2Fe-2S ferredoxins.

Chlorosomes of the green sulfur bacterium Chlorobium tepidum have previously been shown to contain at least 10 polypeptides [Chung, S., Frank, G., Zuber, H., and Bryant, D. A. (1994) Photosynth. Res. 41, 261-275]. Based upon the N-terminal amino acid sequences determined for two of these proteins, the corresponding genes were isolated using degenerate oligonucleotide hybridization probes. The csmI and csmJ genes encode proteins of 244 and 225 amino acids, respectively. A third gene, denoted csmX, that predicts a protein of 221 amino acids with strong sequence similarity to CsmI and CsmJ, was found to be encoded immediately upstream from the csmJ gene. All three proteins have strong sequence similarity in their amino-terminal domains to [2Fe-2S] ferredoxins of the adrenodoxin/putidaredoxin subfamily of ferredoxins. CsmI and CsmJ were overproduced in Escherichia coli, and both proteins were shown by EPR spectroscopy to contain iron-sulfur clusters. The g-tensor and relaxation properties are consistent with their assignment as [2Fe-2S] clusters. Isolated chlorosomes were also shown to contain [2Fe-2S] clusters whose properties were similar to those of the recombinant CsmI and CsmJ proteins. Redox titration of isolated chlorosomes showed these clusters to have potentials of about -201 and +92 mV vs SHE. The former potential is similar to that measured by redox titration of the clusters in inclusion bodies of CsmJ. Possible roles for these iron-sulfur proteins in electron transport and light harvesting are discussed.

Paramagnetic 1H NMR spectroscopy of the reduced, unbound photosystem I subunit PsaC: sequence-specific assignment of contact-shifted resonances and identification of mixed- and equal-valence Fe-Fe pairs in [4Fe-4S] centers FA- and FB-.

The PsaC subunit of Photosystem I (PS I) is a 9.3-kDa protein that binds two important cofactors in photosynthetic electron transfer: the [4Fe-4S] clusters FA and FB. The g-tensor orientation of FA- and FB- is believed to be correlated to the preferential localization of the mixed-valence and equal-valence (ferrous) iron pairs in each [4Fe-4S]+ cluster. The preferential position of the mixed-valence and equal-valence pairs, in turn. can be inferred from the study of the temperature dependence of contact-shifted resonances by 1H NMR spectroscopy. For this, a sequence-specific assignment of these signals is required. The 1H NMR spectrum of reduced, unbound PsaC from Synechococcus sp. PCC 7002 at 280.4 K in 99% D2O solution shows 18 hyperfine-shifted resonances. The non-solvent-exchangeable, hyperfine-shifted resonances of reduced PsaC are clearly identified as belonging to the cysteines coordinating the clusters FA- and FB- by their downfield chemical shifts, by their temperature dependencies, and by their short T1 relaxation times. The usual fast method of assigning the 1H NMR spectra of reduced [4Fe-4S] proteins through magnetization transfer from the oxidized to the reduced state was not feasible in the case of reduced PsaC. Therefore, a de novo self-consistent sequence-specific assignment of the hyperfine-shifted resonances was obtained based on dipolar connectivities from 1D NOE difference spectra and on longitudinal relaxation times using the X-ray structure of Clostridium acidi urici 2[4Fe-4S] cluster ferredoxin at 0.94 A resolution as a model. The results clearly show the same sequence-specific distribution of Curie and anti-Curie cysteines for unbound, reduced PsaC as established for other [4Fe-4S]-containing proteins; therefore, the mixed-valence and equal-valence (ferrous) Fe-Fe pairs in FA- and FB- have the same preferential positions relative to the protein. The analysis reveals that the magnetic properties of the two [4Fe-4S] clusters are essentially indistinguishable in unbound PsaC, in contrast to the PsaC that is bound as a component of the PS I complex.

Recruitment of a foreign quinone into the A(1) site of photosystem I. I. Genetic and physiological characterization of phylloquinone biosynthetic pathway mutants in Synechocystis sp. pcc 6803.

Genes encoding enzymes of the biosynthetic pathway leading to phylloquinone, the secondary electron acceptor of photosystem (PS) I, were identified in Synechocystis sp. PCC 6803 by comparison with genes encoding enzymes of the menaquinone biosynthetic pathway in Escherichia coli. Targeted inactivation of the menA and menB genes, which code for phytyl transferase and 1,4-dihydroxy-2-naphthoate synthase, respectively, prevented the synthesis of phylloquinone, thereby confirming the participation of these two gene products in the biosynthetic pathway. The menA and menB mutants grow photoautotrophically under low light conditions (20 microE m(-2) s(-1)), with doubling times twice that of the wild type, but they are unable to grow under high light conditions (120 microE m(-2) s(-1)). The menA and menB mutants grow photoheterotrophically on media supplemented with glucose under low light conditions, with doubling times similar to that of the wild type, but they are unable to grow under high light conditions unless atrazine is present to inhibit PS II activity. The level of active PS II per cell in the menA and menB mutant strains is identical to that of the wild type, but the level of active PS I is about 50-60% that of the wild type as assayed by low temperature fluorescence, P700 photoactivity, and electron transfer rates. PS I complexes isolated from the menA and menB mutant strains contain the full complement of polypeptides, show photoreduction of F(A) and F(B) at 15 K, and support 82-84% of the wild type rate of electron transfer from cytochrome c(6) to flavodoxin. HPLC analyses show high levels of plastoquinone-9 in PS I complexes from the menA and menB mutants but not from the wild type. We propose that in the absence of phylloquinone, PS I recruits plastoquinone-9 into the A(1) site, where it functions as an efficient cofactor in electron transfer from A(0) to the iron-sulfur clusters.