RESUMEN

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.

Asunto(s)

Chromatiaceae/genética , Chromatiaceae/metabolismo , Lagos/microbiología , Azufre/metabolismo , Colombia Británica , Genoma Bacteriano , Datos de Secuencia Molecular , Oxidación-Reducción , Filogeografía , Análisis de Secuencia de ADN

RESUMEN

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.

Asunto(s)

Biodiversidad , Clorofila/metabolismo , Cianobacterias/clasificación , Cianobacterias/aislamiento & purificación , Manantiales de Aguas Termales/microbiología , Procesos Fototróficos , Proteínas Bacterianas/genética , Clorofila/genética , Análisis por Conglomerados , Cianobacterias/genética , Cianobacterias/fisiología , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Metabolismo Energético , Genes Bacterianos , Variación Genética , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Wyoming

RESUMEN

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.

Asunto(s)

Vías Biosintéticas/genética , Carotenoides/biosíntesis , Chromatiaceae/enzimología , Chromatiaceae/genética , Biomarcadores/metabolismo , Chlorobi/genética , Chromatiaceae/metabolismo , Clonación Molecular , ADN Bacteriano/química , ADN Bacteriano/genética , Escherichia coli/genética , Expresión Génica , Liasas Intramoleculares/genética , Metiltransferasas/genética , Datos de Secuencia Molecular , Oxidorreductasas/genética , Filogenia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido

RESUMEN

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.

Asunto(s)

Proteínas Bacterianas/química , Carotenoides/biosíntesis , Chlorobium/metabolismo , Chlorobium/ultraestructura , Complejos de Proteína Captadores de Luz/química , Proteínas Bacterianas/genética , Fenómenos Biofísicos , Biofisica , Chlorobium/genética , Microscopía por Crioelectrón , Genes Bacterianos , Complejos de Proteína Captadores de Luz/genética , Mutación , Difracción de Rayos X

RESUMEN

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.

Asunto(s)

Oxígeno/metabolismo , Plastocianina/química , Secuencia de Aminoácidos , Clonación Molecular , Cobre/metabolismo , Cianobacterias/metabolismo , Transporte de Electrón , Escherichia coli/metabolismo , Iones , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Plastocianina/genética , Conformación Proteica , Factores de Tiempo

RESUMEN

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.

Asunto(s)

Chlorobi/genética , Cromosomas Bacterianos , Silenciador del Gen , Fijación del Nitrógeno/genética , Transformación Bacteriana , Farmacorresistencia Microbiana/genética , Genes Bacterianos , Marcadores Genéticos , Recombinación Genética , Selección Genética

RESUMEN

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.

Asunto(s)

Cianobacterias/genética , Complejos de Proteína Captadores de Luz , Ficocianina/genética , Proteínas Bacterianas/metabolismo , Clorofila/química , Clorofila/metabolismo , Cianobacterias/metabolismo , Cianobacterias/ultraestructura , Luz , Proteínas de la Membrana/metabolismo , Mutación , Oxígeno/metabolismo , Fotosíntesis , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Ficobilisomas , Ficocianina/análisis , Espectrometría de Fluorescencia , Temperatura

RESUMEN

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.

Asunto(s)

Proteínas Bacterianas/metabolismo , Chlorobi/metabolismo , Ferredoxinas/metabolismo , Membranas Intracelulares/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Chlorobi/química , Chlorobi/genética , Clonación Molecular , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Escherichia coli/genética , Ferredoxinas/biosíntesis , Ferredoxinas/química , Ferredoxinas/genética , Genes Bacterianos , Membranas Intracelulares/química , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/metabolismo , Datos de Secuencia Molecular , Oxidación-Reducción , Plásmidos/síntesis química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido

RESUMEN

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.

Asunto(s)

Cianobacterias/química , Proteínas Hierro-Azufre/química , Hierro/química , Proteínas de la Membrana , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Complejo de Proteína del Fotosistema I , Proteínas/química , Proteínas Recombinantes/química , Cianobacterias/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Proteínas Hierro-Azufre/metabolismo , Espectroscopía de Resonancia Magnética , Magnetismo , Modelos Estructurales , Oxidación-Reducción , Proteínas/metabolismo , Temperatura

RESUMEN

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.

Asunto(s)

Cianobacterias/metabolismo , Proteínas de Escherichia coli , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Vitamina K 1/biosíntesis , Transferasas Alquil y Aril/genética , Clorofila/metabolismo , Cianobacterias/genética , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Flavodoxina/metabolismo , Genes Bacterianos , Hidroliasas/genética , Membranas Intracelulares , Proteínas Hierro-Azufre/metabolismo , Luz , Complejos de Proteína Captadores de Luz , Mutación , Fenotipo , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/efectos de la radiación , Complejo de Proteína del Fotosistema I

RESUMEN

The nrtP and narB genes, encoding nitrate/nitrite permease and nitrate reductase, respectively, were isolated from the marine cyanobacterium Synechococcus sp. strain PCC 7002 and characterized. NrtP is a member of the major facilitator superfamily and is unrelated to the ATP-binding cassette-type nitrate transporters that previously have been described for freshwater strains of cyanobacteria. However, NrtP is similar to the NRT2-type nitrate transporters found in diverse organisms. An nrtP mutant strain consumes nitrate at a 4.5-fold-lower rate than the wild type, and this mutant grew exponentially on a medium containing 12 mM nitrate at a rate approximately 2-fold lower than that of the wild type. The nrtP mutant cells could not consume nitrite as rapidly as the wild type at pH 10, suggesting that NrtP also functions in nitrite uptake. A narB mutant was unable to grow on a medium containing nitrate as a nitrogen source, although this mutant could grow on media containing urea or nitrite with rates similar to those of the wild type. Exogenously added nitrite enhanced the in vivo activity of nitrite reductase in the narB mutant; this suggests that nitrite acts as a positive effector of nitrite reductase. Transcripts of the nrtP and narB genes were detected in cells grown on nitrate but were not detected in cells grown on urea or ammonia. Transcription of the nrtP and narB genes is probably controlled by the NtcA transcription factor for global nitrogen control. The discovery of a nitrate/nitrite permease in Synechococcus sp. strain PCC 7002 suggests that significant differences in nutrient transporters may occur in marine and freshwater cyanobacteria.

Asunto(s)

Proteínas Bacterianas/metabolismo , Cianobacterias/enzimología , Proteínas de Transporte de Membrana/metabolismo , Nitratos/metabolismo , Nitritos/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Cianobacterias/genética , Regulación Bacteriana de la Expresión Génica , Proteínas de Transporte de Membrana/genética , Datos de Secuencia Molecular , Mutación , Nitrato-Reductasa , Nitrato Reductasas/genética , Mapeo Físico de Cromosoma , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Factores de Tiempo , Transcripción Genética

RESUMEN

PsaE is a small basic subunit located on the stromal (cytoplasmic) side of photosystem I. In cyanobacteria, this subunit participates in cyclic electron transport and modulates the interactions of the complex with soluble ferredoxin. The PsaE protein isolated from the cyanobacterium Synechococcus sp. strain PCC 7002 adopts the beta topology of an SH3 domain, with five beta strands (betaA through betaE) and a turn of 3(10) helix between strands betaD and betaE [Falzone, C. J., Kao, Y.-H., Zhao, J., Bryant, D. A., and Lecomte, J. T. J. (1994) Biochemistry 33, 6052-6062]. The primary structure of the PsaE protein is strongly conserved across all oxygen-evolving photosynthetic organisms. However, variability in loop lengths, as well as N- or C-terminal extensions, suggests that the structure of a second representative PsaE subunit would be useful to characterize the interactions among photosystem I polypeptides. In this work, the solution structure of PsaE from the cyanobacterium Nostoc sp. strain PCC 8009 was determined by NMR methods. Compared to PsaE from Synechococcus sp. strain PCC 7002, this PsaE has a seven-residue deletion in the loop connecting strands betaC and betaD, and an eight-residue C-terminal extension. Angular and distance restraints derived from homonuclear and heteronuclear NMR experiments were used to calculate structures by a distance-geometry/simulated-annealing protocol. A family of 20 structures (rmsd of 0.24 A in the regular secondary structure) is presented. Differences between the two cyanobacterial proteins are mostly confined to the CD loop region; the C-terminal extension is disordered. The thermodynamic stability of Nostoc sp. strain PCC 8009 PsaE toward urea denaturation was measured by circular dichroism and fluorescence spectroscopy, and thermal denaturation was monitored by UV absorption spectroscopy. Chemical and thermal denaturation curves are modeled satisfactorily with two-state processes. The DeltaG degrees of unfolding at room temperature is 12.4 +/- 0.3 kJ mol(-1) (pH 5), and the thermal transition midpoint is 59 +/- 1 degrees C (pH 7). Interactions with other proteins in the photosystem I complex may aid in maintaining PsaE in its native state under physiological conditions.

Asunto(s)

Cianobacterias/química , Fragmentos de Péptidos/química , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Complejo de Proteína del Fotosistema I , Secuencia de Aminoácidos , Cristalografía por Rayos X , Calor , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/aislamiento & purificación , Proteínas del Complejo del Centro de Reacción Fotosintética/aislamiento & purificación , Desnaturalización Proteica , Homología de Secuencia de Aminoácido , Soluciones , Espectrometría de Fluorescencia , Termodinámica , Urea

RESUMEN

The effect of low temperature on cell growth, photosynthesis, photoinhibition, and nitrate assimilation was examined in the cyanobacterium Synechococcus sp. PCC 6301 to determine the factor that limits growth. Synechococcus sp. PCC 6301 grew exponentially between 20 degreesC and 38 degreesC, the growth rate decreased with decreasing temperature, and growth ceased at 15 degreesC. The rate of photosynthetic oxygen evolution decreased more slowly with temperature than the growth rate, and more than 20% of the activity at 38 degreesC remained at 15 degreesC. Oxygen evolution was rapidly inactivated at high light intensity (3 mE m-2 s-1) at 15 degreesC. Little or no loss of oxygen evolution was observed under the normal light intensity (250 microE m-2 s-1) for growth at 15 degreesC. The decrease in the rate of nitrate consumption by cells as a function of temperature was similar to the decrease in the growth rate. Cells could not actively take up nitrate or nitrite at 15 degreesC, although nitrate reductase and nitrite reductase were still active. These data demonstrate that growth at low temperature is not limited by a decrease in the rate of photosynthetic electron transport or by photoinhibition, but that inactivation of the nitrate/nitrite transporter limits growth at low temperature.

Asunto(s)

Proteínas de Transporte de Anión , Cianobacterias/metabolismo , Ácido Nítrico/metabolismo , Proteínas Bacterianas/metabolismo , Transporte Biológico Activo , Carbono/metabolismo , Proteínas Portadoras/metabolismo , División Celular , Frío , Cianobacterias/crecimiento & desarrollo , Cianobacterias/efectos de la radiación , Luz , Lípidos de la Membrana/metabolismo , Nitrato-Reductasa , Nitrato Reductasas/metabolismo , Transportadores de Nitrato , Nitrito Reductasas/metabolismo , Nitrógeno/metabolismo , Ácido Nitroso/metabolismo , Oxígeno/metabolismo , Fotosíntesis

RESUMEN

Flavodoxin can function as an alternative electron acceptor for photosystem I (PSI) in place of ferredoxin under iron-limiting conditions. The isiB gene, encoding the flavodoxin in Synechococcus sp. PCC 7002, was overexpressed in Escherichia coli. Under the conditions employed, most recombinant flavodoxin (rFlvd) was in soluble form with cofactor correctly inserted. The absorption spectrum of rFlvd was identical to that of the native flavodoxin of the cyanobacteria. Photoreduction of rFlvd by PSI particles and thylakoid membranes was determined directly by monitoring the absorption change at 467 nm. The optimal conditions for rFlvd photoreduction were determined. Compared to other methods currently employed to measure PSI activity such as oxygen uptake in the presence of methyl viologen and NADP+ photoreduction in the presence of ferredoxin and ferredoxin:NADP+ oxidoreductase, measurement of PSI activity with flavodoxin as an electron acceptor has several advantages. It measures the full-chain electron transfer chain of PSI since flavodoxin accepts electrons from FA/FB and it is much simpler than the method with NADP+ photoreduction. With this method, we found that the affinity of wild-type PSI for rFlvd was 35% higher than that of the PsaE-less PSI, showing that this method is sensitive to structural changes of PSI. Our results demonstrate that rFlvd photoreduction is an effective and simple method for PSI activity measurement.

Asunto(s)

Proteínas Bacterianas/metabolismo , Cianobacterias/metabolismo , Flavodoxina/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Complejo de Proteína del Fotosistema I , Proteínas Bacterianas/genética , Reactivos de Enlaces Cruzados , Cianobacterias/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Flavodoxina/genética , Concentración de Iones de Hidrógeno , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/fisiología , Isopropil Tiogalactósido/farmacología , Cinética , Magnesio/farmacología , Mutación , Oxidación-Reducción/efectos de los fármacos , Fotosíntesis/efectos de los fármacos , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Análisis Espectral , Urea/farmacología

RESUMEN

The group 1 and group 2 sigma70-type sigma factors of the green sulfur bacterium Chlorobium tepidum and of the green nonsulfur bacterium Chloroflexus aurantiacus were cloned and characterized. Cb. tepidum was found to contain one sigma70-type sigma factor; the expression of the gene was analyzed by Northern blot hybridization and primer-extension mapping. Cf. aurantiacus has genes encoding four sigma factors of groups 1 and 2. The expression of these genes was examined in cells grown aerobically and anaerobically. The sigC gene was expressed at approximately equal levels under both conditions, resulting in its designation as the group 1 sigma factor of this organism. The only other detectable transcripts arose from the sigB gene, which was expressed at higher levels during aerobic growth. A phylogenetic tree was obtained using the group 1 sigma factors of Cb. tepidum, Cf. aurantiacus, and diverse eubacteria as the molecular marker. The resulting phylogenetic tree shows that Cb. tepidum and Cf. aurantiacus are related to each other and to the cyanobacteria. The relationship of the group 2 sigma factors of Cf. aurantiacus and the cyanobacteria was more specifically examined phylogenetically. The group 2 sigma factors of Cf. aurantiacus probably arose by gene duplication events after the split of the green nonsulfur bacteria from other photosynthetic eubacteria.

Asunto(s)

Proteínas Bacterianas/genética , Chlorobi/genética , Factor sigma/genética , Secuencia de Bases , Chlorobi/clasificación , Datos de Secuencia Molecular , Filogenia , ARN Bacteriano/genética

RESUMEN

PsaD is a small, extrinsic polypeptide located on the stromal side (cytoplasmic side in cyanobacteria) of the photosystem I reaction centre complex. The gene from the cyanobacterium Nostoc sp. PCC 8009 was expressed in Escherichia coli and the structure of the recovered protein in solution investigated. Size-exclusion chromatography, dynamic light scattering and measurement of 15N transverse relaxation times showed that the protein is a stable dimer in solution, whereas in the reaction centre complex it is a monomer. NMR experiments showed that the dimer is symmetrical and that there are at least two domains, one structured and the remainder unstructured. The structured domain contains a small amount of beta-sheet. Three-dimensional heteronuclear NMR spectra of [13C, 15N]PsaD showed that the structured domain is associated with the central part of the sequence while the N- and C-terminal regions are mobile. Evidence was obtained for a shift in equilibrium between two slightly different conformational states at about pH 6, and the protein was shown to bind to PsaE preferentially at neutral pH. Addition of trifluoroethanol was shown to induce the formation of a small amount of alpha-helix, and the form present in 30% trifluoroethanol appears to be more closely related to the in situ structure, which has been reported to contain one short helix in crystals [Schubert, W.-D., Klukas, O., Krauss, N., Saenger, W., Fromme, P. & Witt, H. T. (1997) J. Mol. Biol. 272, 741-769]. The significance of these findings for the assembly of the complex is discussed.

Asunto(s)

Proteínas del Complejo del Centro de Reacción Fotosintética/química , Complejo de Proteína del Fotosistema I , Proteínas de Plantas/química , Secuencia de Aminoácidos , Dicroismo Circular , Cianobacterias , Dimerización , Luz , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Dispersión de Radiación , Soluciones

RESUMEN

Targeted mutagenesis was used to investigate the roles of the CsmA and CsmC proteins of the chlorosomes of the green bacteria Chlorobium tepidum and Chlorobium vibrioforme 8327. Under the photoautotrophic growth conditions employed, CsmA is required for the viability of the cells but CsmC is dispensable. The absence of CsmC caused a small red shift in the near-infrared absorption maximum of bacteriochlorophyll d in whole cells and chlorosomes, but chlorosomes were assembled in and could be isolated from the csmC mutant. The doubling time of the csmC mutant was approximately twice that of the wild-type strain. Fluorescence emission measurements suggested that energy transfer from the bulk bacteriochlorophyll d to another pigment, perhaps bacteriochlorophyll a, emitting at 800-804 nm, was less efficient in the csmC mutant cells than in wild-type cells. These studies establish that transformation and homologous recombination can be employed in targeted mutagenesis of Chlorobium sp. and further demonstrate that chlorosome proteins play important roles in the structure and function of these light-harvesting organelles.

Asunto(s)

Proteínas Bacterianas/fisiología , Chlorobi/genética , Chlorobi/fisiología , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Proteínas Bacterianas/genética , Genes Bacterianos/genética , Complejos de Proteína Captadores de Luz , Mutagénesis Insercional , Orgánulos/química , Orgánulos/fisiología , Recombinación Genética , Espectrometría de Fluorescencia , Transformación Bacteriana

RESUMEN

Laboratory conditions have been identified that cause the rapid death of cultures of cyanobacteria producing urease. Once the death phase had initiated in the stationary growth phase, cells were rapidly bleached of all pigmentation. Null mutations in the ureC gene, encoding the alpha subunit of urease, were constructed, and these mutants were no longer sensitive to growth in the presence of urea. High levels of peroxides, including lipid peroxides, were detected in the bleaching cells. Exogenously added polyunsaturated fatty acids triggered a similar death response. Vitamin E suppressed the formation of peroxides and delayed the onset of cell bleaching. The results suggest that these cyanobacterial cells undergo a metabolic imbalance that ultimately leads to oxidative stress and lipid peroxide formation. These observations may provide insights into the mechanism of sudden cyanobacterial bloom disappearance in nature.

Asunto(s)

Cianobacterias/efectos de los fármacos , Urea/farmacología , Ureasa/biosíntesis , Cianobacterias/crecimiento & desarrollo , Cianobacterias/metabolismo , Ácidos Grasos Insaturados/farmacología , Eliminación de Gen , Genes Bacterianos/genética , Herbicidas/farmacología , Peroxidación de Lípido , Paraquat/farmacología , Ureasa/genética

RESUMEN

Using RecA as the phylogenetic marker, the relationships of the green sulfur bacterium Chlorobium tepidum and the green non-sulfur bacterium Chloroflexus aurantiacus to other eubacteria were investigated. The recA genes of the two organisms were cloned, and the resulting protein sequences aligned with 86 other eubacterial RecA sequences. Cb. tepidum was placed as the nearest relative to the Cytophaga/Flexibacter/Bacteriodes group, a relationship supported by results obtained with several phylogenetic markers. Cf. aurantiacus was placed near Chlamydia trachomatis and the high-GC Gram-positives; however, this branching pattern was not strongly supported statistically by bootstrap analyses. Possible reasons for this ambiguity are discussed.

Asunto(s)

Chlorobi/genética , Filogenia , Rec A Recombinasas/genética , Secuencia de Aminoácidos , Clonación Molecular , Genes Bacterianos/genética , Datos de Secuencia Molecular , Mapeo Restrictivo , Alineación de Secuencia , Análisis de Secuencia de ADN

RESUMEN

The sigD and sigE genes, which encode two alternative sigma-factors from the unicellular marine cyanobacterium Synechococcus sp. PCC 7002, were cloned and characterized. Strains in which the sigD and sigE genes were insertionally inactivated were viable under standard laboratory conditions, indicating that SigD and SigE are group 2 sigma-factors. When stationary-phase cells were diluted into fresh growth medium, it was observed that the sigE mutant strain required longer times to re-establish exponential growth than the wild-type strain. By monitoring the growth rates in such dilution experiments, it was observed that the lag times for the mutant strain became progressively longer as the original cultures progressed towards stationary phase. Transcripts for the sigE gene initially increased and subsequently decreased as cells grew further into stationary phase. It was determined that a functional SigE protein is required for the expression of the starvation-induced protein DpsA/PexB. The results suggest that SigE is involved in the transcription of genes specifically expressed in the post-exponential phase.

Asunto(s)

Proteínas Bacterianas/genética , Cianobacterias/genética , Flagelina/genética , Genes Bacterianos/genética , Factor sigma/genética , Transcripción Genética , Secuencia de Aminoácidos , Proteínas Bacterianas/aislamiento & purificación , Cianobacterias/química , Flagelina/aislamiento & purificación , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Mutagénesis , ARN Mensajero/análisis , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factor sigma/aislamiento & purificación