RESUMO
The largest light-harvesting antenna in nature, the chlorosome, is a heterogeneous helical BChl self-assembly that has evolved in green bacteria to harvest light for performing photosynthesis in low-light environments. Guided by NMR chemical shifts and distance constraints for Chlorobaculum tepidum wild-type chlorosomes, the two contrasting packing modes for syn-anti parallel stacks of BChl c to form polar 2D arrays, with dipole moments adding up, are explored. Layered assemblies were optimized using local orbital density functional and plane wave pseudopotential methods. The packing mode with the lowest energy contains syn-anti and anti-syn H-bonding between stacks. It can accommodate R and S epimers, and side chain variability. For this packing, a match with the available EM data on the subunit axial repeat and optical data is obtained with multiple concentric cylinders for a rolling vector with the stacks running at an angle of 21° to the cylinder axis and with the BChl dipole moments running at an angle ß â¼ 55° to the tube axis, in accordance with optical data. A packing mode involving alternating syn and anti parallel stacks that is at variance with EM appears higher in energy. A weak cross-peak at -6 ppm in the MAS NMR with 50 kHz spinning, assigned to C-181, matches the shift of antiparallel dimers, which possibly reflects a minor impurity-type fraction in the self-assembled BChl c.
Assuntos
Chlorobi , Chlorobi/metabolismo , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismo , Espectroscopia de Ressonância Magnética , Modelos MolecularesRESUMO
Three strains of thermophilic green sulfur bacteria (GSB) are known; all are from microbial mats in hot springs in Rotorua, New Zealand (NZ) and belong to the species Chlorobaculum tepidum. Here, we describe diverse populations of GSB inhabiting Travel Lodge Spring (TLS) (NZ) and hot springs ranging from 36.1 °C to 51.1 °C in the Republic of the Philippines (PHL) and Yellowstone National Park (YNP), Wyoming, USA. Using targeted amplification and restriction fragment length polymorphism analysis, GSB 16S rRNA sequences were detected in mats in TLS, one PHL site, and three regions of YNP. GSB enrichments from YNP and PHL mats contained small, green, nonmotile rods possessing chlorosomes, chlorobactene, and bacteriochlorophyll c. Partial 16S rRNA gene sequences from YNP, NZ, and PHL mats and enrichments from YNP and PHL samples formed distinct phylogenetic clades, suggesting geographic isolation, and were associated with samples differing in temperature and pH, suggesting adaptations to these parameters. Sequences from enrichments and corresponding mats formed clades that were sometimes distinct, increasing the diversity detected. Sequence differences, monophyly, distribution patterns, and evolutionary simulation modeling support our discovery of at least four new putative moderately thermophilic Chlorobaculum species that grew rapidly at 40 °C to 44 °C.
RESUMO
Light energy absorption and transfer are very important processes in photosynthesis. In green sulfur bacteria light is absorbed primarily by the chlorosomes and its energy is transferred via the Fenna-Matthews-Olson (FMO) proteins to a homodimeric reaction center (RC). Here, we report the cryogenic electron microscopic structure of the intact FMO-RC apparatus from Chlorobaculum tepidum at 2.5 Å resolution. The FMO-RC apparatus presents an asymmetric architecture and contains two FMO trimers that show different interaction patterns with the RC core. Furthermore, the two permanently bound transmembrane subunits PscC, which donate electrons to the special pair, interact only with the two large PscA subunits. This structure fills an important gap in our understanding of the transfer of energy from antenna to the electron transport chain of this RC and the transfer of electrons from reduced sulfur compounds to the special pair.
Assuntos
Chlorobi , Complexo de Proteínas do Centro de Reação Fotossintética , Complexo de Proteínas do Centro de Reação Fotossintética/química , Chlorobi/metabolismo , Microscopia Crioeletrônica , Proteínas de Bactérias/metabolismo , Enxofre/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismoRESUMO
Light harvesting and charge separation are functions of chlorophyll and bacteriochlorophyll pigments. While most photosynthetic organisms use (bacterio)chlorophylls with a phytyl (2-phytenyl) group as the hydrophobic isoprenoid tail, Halorhodospira halochloris, an anoxygenic photosynthetic bacterium belonging to Gammaproteobacteria, produces bacteriochlorophylls with a unique 6,7,14,15-tetrahydrogeranylgeranyl (2,10-phytadienyl) tail. Geranylgeranyl reductase (GGR), encoded by the bchP gene, catalyzes hydrogenation at three unsaturated C=C bonds of a geranylgeranyl group, giving rise to the phytyl tail. In this study, we discovered that H. halochloris GGR exhibits only partial hydrogenation activities, resulting in the tetrahydrogeranylgeranyl tail formation. We hypothesized that the hydrogenation activity of H. halochloris GGR differed from that of Chlorobaculum tepidum GGR, which also produces a pigment with partially reduced hydrophobic tails (2,6-phytadienylated chlorophyll a). An engineered GGR was also constructed and demonstrated to perform only single hydrogenation, resulting in the dihydrogeranylgeranyl tail formation. H. halochloris original and variant GGRs shed light on GGR catalytic mechanisms and offer prospective bioengineering tools in the microbial production of isoprenoid compounds. IMPORTANCE Geranylgeranyl reductase (GGR) catalyzes the hydrogenation of carbon-carbon double bonds of unsaturated hydrocarbons of isoprenoid compounds, including α-tocopherols, phylloquinone, archaeal cell membranes, and (bacterio)chlorophyll pigments in various organisms. GGRs in photosynthetic organisms, including anoxygenic phototrophic bacteria, cyanobacteria, and plants perform successive triple hydrogenation to produce chlorophylls and bacteriochlorophylls with a phytyl chain. Here, we demonstrated that the GGR of a gammaproteobacterium Halorhodospira halochloris catalyzed unique double hydrogenation to produce bacteriochlorophylls with a tetrahydrogeranylgeranyl tail. We also constructed a variant enzyme derived from H. halochloris GGR that performs only single hydrogenation. The results of this study provide new insights into catalytic mechanisms of multiposition reductions by a single enzyme.
Assuntos
Bacterioclorofilas , Chlorobi , Bacterioclorofilas/química , Carbono , Chlorobi/metabolismo , Clorofila/química , Clorofila/metabolismo , Clorofila A , Ectothiorhodospiraceae , Hidrogenação , Oxirredutases , Estudos Prospectivos , Proteobactérias/metabolismo , TerpenosRESUMO
2-Oxoglutarate:ferredoxin oxidoreductase from Chlorobaculum tepidum (CtOGOR) is a carbon-fixing enzyme in the reductive TCA cycle that reversibly carboxylates succinyl-CoA to yield 2-oxoglutarate. CtOGOR is a heterotetramer of two large (α = 68 kDa) and two small (ß = 38 kDa) subunits. The αß protomer harbors one thiamine pyrophosphate and two 4Fe-4S clusters. Nonetheless, the enzyme has a considerable oxygen tolerance with a half-life of 143 min at 215 µM dissolved oxygen. Kinetic analyses of the purified recombinant CtOGOR revealed a lower Km for succinyl-CoA than for 2-oxoglutarate. Cellular levels of 2-oxoglutarate and glutamatea product of glutamine oxoglutarate aminotransferase and glutamate dehydrogenaseincreased more than twofold in the exponential phase compared with the control strain, leading to an approximately >30% increase in the photoautotrophic growth rate. Thus, CtOGOR was successfully produced in Synechocystis, thereby boosting carboxylation, resulting in enhanced photoautotrophic growth.
RESUMO
The trimeric nature of the Fenna-Matthews-Olson (FMO) protein antenna complex from green sulfur phototrophic bacteria was investigated. Mutations were introduced into the protein at positions 142 and 198, which were chosen to destabilize the intra-trimer salt bridges between adjacent monomers. Strains bearing the mutations R142L, R198L, or their combination, exhibited altered optical absorption spectra of purified membranes and fluoresced more intensely than the wild type. In particular, the introduction of the R142L mutation resulted in slower culture growth rates, as well as an FMO complex that was not able to be isolated in appreciable quantities, while the R198L mutation yielded an FMO complex with increased sensitivity to sodium thiocyanate and Triton X-100 treatments. Native and denaturing PAGE experiments suggest that much of the FMO complexes in the mutant strains pool with the insoluble material upon membrane solubilization with n-dodecyl ß-D-maltoside, a mild nonionic detergent. Taken together, our results suggest that the quaternary structure of the FMO complex, the homotrimer, is an important factor in the maintenance of the complex's tertiary structure.
Assuntos
Proteínas de Bactérias/química , Bacterioclorofilas/química , Chlorobi/química , Complexos de Proteínas Captadores de Luz/química , Estrutura Quaternária de Proteína , Substituição de Aminoácidos , Membrana Celular/efeitos da radiação , Chlorobi/efeitos da radiação , Modelos Moleculares , Complexos Multiproteicos , Mutação , Fotossíntese , Estabilidade ProteicaRESUMO
Low-molecular-weight (LMW) thiols mediate redox homeostasis and the detoxification of chemical stressors. Despite their essential functions, the distribution of LMW thiols across cellular life has not yet been defined. LMW thiols are also thought to play a central role in sulfur oxidation pathways in phototrophic bacteria, including the Chlorobiaceae Here we show that Chlorobaculum tepidum synthesizes a novel LMW thiol with a mass of 412 ± 1 Da corresponding to a molecular formula of C14H24N2O10S, which suggests that the new LMW thiol is closely related to bacillithiol (BSH), the major LMW thiol of low-G+C Gram-positive bacteria. The Cba. tepidum LMW thiol structure was N-methyl-bacillithiol (N-Me-BSH), methylated on the cysteine nitrogen, the fourth instance of this modification in metabolism. Orthologs of bacillithiol biosynthetic genes in the Cba. tepidum genome and the CT1040 gene product, N-Me-BSH synthase, were required for N-Me-BSH synthesis. N-Me-BSH was found in all Chlorobiaceae examined as well as Polaribacter sp. strain MED152, a member of the Bacteroidetes A comparative genomic analysis indicated that BSH/N-Me-BSH is synthesized not only by members of the Chlorobiaceae, Bacteroidetes, Deinococcus-Thermus, and Firmicutes but also by Acidobacteria, Chlamydiae, Gemmatimonadetes, and Proteobacteria. Thus, BSH and derivatives appear to be the most broadly distributed LMW thiols in biology.IMPORTANCE Low-molecular-weight thiols are key metabolites that participate in many basic cellular processes: central metabolism, detoxification, and oxidative stress resistance. Here we describe a new thiol, N-methyl-bacillithiol, found in an anaerobic phototrophic bacterium and identify a gene that is responsible for its synthesis from bacillithiol, the main thiol metabolite in many Gram-positive bacteria. We show that the presence or absence of this gene in a sequenced genome accurately predicts thiol content in distantly related bacteria. On the basis of these results, we analyzed genome data and predict that bacillithiol and its derivatives are the most widely distributed thiol metabolites in biology.
Assuntos
Vias Biossintéticas/genética , Chlorobi/genética , Chlorobi/metabolismo , Cisteína/análogos & derivados , Glucosamina/análogos & derivados , Cisteína/química , Cisteína/metabolismo , Genoma Bacteriano , Glucosamina/química , Glucosamina/metabolismo , Estrutura Molecular , Peso MolecularRESUMO
Chlorobaculum tepidum, a green sulfur bacterium, utilizes chlorobactene as its major carotenoid, and this organism also accumulates a reduced form of this monocyclic pigment, 1',2'-dihydrochlorobactene. The protein catalyzing this reduction is the last unidentified enzyme in the biosynthetic pathways for all of the green sulfur bacterial pigments used for photosynthesis. The genome of C. tepidum contains two paralogous genes encoding members of the FixC family of flavoproteins: bchP, which has been shown to encode an enzyme of bacteriochlorophyll biosynthesis; and bchO, for which a function has not been assigned. Here we demonstrate that a bchO mutant is unable to synthesize 1',2'-dihydrochlorobactene, and when bchO is heterologously expressed in a neurosporene-producing mutant of the purple bacterium, Rhodobacter sphaeroides, the encoded protein is able to catalyze the formation of 1,2-dihydroneurosporene, the major carotenoid of the only other organism reported to synthesize 1,2-dihydrocarotenoids, Blastochloris viridis Identification of this enzyme completes the pathways for the synthesis of photosynthetic pigments in Chlorobiaceae, and accordingly and consistent with its role in carotenoid biosynthesis, we propose to rename the gene cruI Notably, the absence of cruI in B. viridis indicates that a second 1,2-carotenoid reductase, which is structurally unrelated to CruI (BchO), must exist in nature. The evolution of this carotenoid reductase in green sulfur bacteria is discussed herein.
Assuntos
Bacterioclorofilas/biossíntese , Carotenoides/biossíntese , Chlorobi/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bacterioclorofilas/química , Bacterioclorofilas/genética , Vias Biossintéticas/genética , Carotenoides/química , Carotenoides/genética , Carotenoides/metabolismo , Chlorobi/química , Chlorobium/enzimologia , Chlorobium/genética , Genoma Bacteriano/genética , Oxirredutases/química , Oxirredutases/genética , Fotossíntese/genéticaRESUMO
The green sulfur bacteria (Chlorobiaceae) are anaerobes that use electrons from reduced sulfur compounds (sulfide, S0, and thiosulfate) as electron donors for photoautotrophic growth. Chlorobaculum tepidum, the model system for the Chlorobiaceae, both produces and consumes extracellular S0 globules depending on the availability of sulfide in the environment. These physiological changes imply significant changes in gene regulation, which has been observed when sulfide is added to Cba. tepidum growing on thiosulfate. However, the underlying mechanisms driving these gene expression changes, i.e., the specific regulators and promoter elements involved, have not yet been defined. Here, differential RNA sequencing (dRNA-seq) was used to globally identify transcript start sites (TSS) that were present during growth on sulfide, biogenic S0, and thiosulfate as sole electron donors. TSS positions were used in combination with RNA-seq data from cultures growing on these same electron donors to identify both basal promoter elements and motifs associated with electron donor-dependent transcriptional regulation. These motifs were conserved across homologous Chlorobiaceae promoters. Two lines of evidence suggest that sulfide-mediated repression is the dominant regulatory mode in Cba. tepidum First, motifs associated with genes regulated by sulfide overlap key basal promoter elements. Second, deletion of the Cba. tepidum1277 (CT1277) gene, encoding a putative regulatory protein, leads to constitutive overexpression of the sulfide:quinone oxidoreductase CT1087 in the absence of sulfide. The results suggest that sulfide is the master regulator of sulfur metabolism in Cba. tepidum and the Chlorobiaceae Finally, the identification of basal promoter elements with differing strengths will further the development of synthetic biology in Cba. tepidum and perhaps other ChlorobiaceaeIMPORTANCE Elemental sulfur is a key intermediate in biogeochemical sulfur cycling. The photoautotrophic green sulfur bacterium Chlorobaculum tepidum either produces or consumes elemental sulfur depending on the availability of sulfide in the environment. Our results reveal transcriptional dynamics of Chlorobaculum tepidum on elemental sulfur and increase our understanding of the mechanisms of transcriptional regulation governing growth on different reduced sulfur compounds. This report identifies genes and sequence motifs that likely play significant roles in the production and consumption of elemental sulfur. Beyond this focused impact, this report paves the way for the development of synthetic biology in Chlorobaculum tepidum and other Chlorobiaceae by providing a comprehensive identification of promoter elements for control of gene expression, a key element of strain engineering.
Assuntos
Chlorobi/genética , Chlorobi/metabolismo , Metabolismo Energético , Regulação Bacteriana da Expressão Gênica , Sulfetos/metabolismo , Enxofre/metabolismo , Oxirredução , Regiões Promotoras Genéticas , RNA/metabolismo , Análise de Sequência de RNA , Compostos de Enxofre/metabolismoRESUMO
Sulfide:quinone oxidoreductase (SQR) is the primary sulfide-oxidizing enzyme found in all three domains of life. Of the six phylogenetically distinct types of SQR, four have representatives that have been biochemically characterized. The genome of Chlorobaculum tepidum encodes three SQR homologs. One of these, encoded by CT1087, is a type VI SQR that has been previously shown to be required for growth at high sulfide concentrations and to be expressed in sulfide-dependent manner. Therefore, CT1087 was hypothesized to be a high sulfide adapted SQR. CT1087 was expressed in Escherichia coli with an N-terminal His-tag (CT1087NHis6) and purified by Ni-NTA chromatography. CT1087NHis6 was active and contained FAD as a strongly bound cofactor. The measured kinetic parameters for CT1087NHis6 indicate a low affinity for sulfide and a high enzymatic turnover rate consistent with the hypothesis for its function inferred from genetic and expression data. These are the first kinetic data for a type VI SQR and have implications for structure-function analyses of all SQR's.
Assuntos
Chlorobi/enzimologia , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Sulfetos/metabolismo , Chlorobi/crescimento & desenvolvimento , Chlorobi/metabolismo , Metabolismo Energético , Escherichia coli/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Cinética , NAD(P)H Desidrogenase (Quinona)/isolamento & purificação , FilogeniaRESUMO
The photosynthetic green sulfur bacterium Chlorobaculum (Cba.) tepidum produces bacteriochlorophyll (BChl) c pigments bearing a chiral 1-hydroxyethyl group at the 3-position, which self-aggregate to construct main light-harvesting antenna complexes, chlorosomes. The secondary alcoholic hydroxy group is requisite for chlorosomal aggregation and biosynthesized by hydrating the 3-vinyl group of their precursors. Using recombinant proteins of Cba. tepidum BchF and BchV, we examined in vitro enzymatic hydration of some 3-vinyl-chlorophyll derivatives. Both the enzymes catalyzed stereoselective hydration of zinc 3-vinyl-8-ethyl-12-methyl-bacteriopheophorbide c or d to the zinc 31 R-bacteriopheophorbide c or d homolog, respectively, with a slight amount of the 31 S-epimric species. A similar R-stereoselectivity was observed in the BchF-hydration of zinc 3-vinyl-8-ethyl- and propyl-12-ethyl-bacteriopheophorbides c, while their BchV-hydration gave a relatively larger amount of the 31 S-epimers. The in vitro stereoselective hydration confirmed the in vivo production of the S-epimeric species by BchV. The enzymatic hydration for the above 8-propylated substrate proceeded more slowly than that for the 8-ethylated, and the 8-isobutylated substrate was no longer hydrated. Based on these results, biosynthetic pathways of BChl c homologs and epimers are proposed.
Assuntos
Proteínas de Bactérias/biossíntese , Bacterioclorofilas/biossíntese , Chlorobi/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bacterioclorofilas/química , Vias Biossintéticas , Chlorobi/enzimologia , Cromatografia Líquida de Alta Pressão , Técnicas In Vitro , Estrutura Molecular , EstereoisomerismoRESUMO
The thermodynamics for the first electron transfer step for sulfide and oxygen indicates that the reaction is unfavorable as unstable superoxide and bisulfide radical ions would need to be produced. However, a two-electron transfer is favorable as stable S(0) and peroxide would be formed, but the partially filled orbitals in oxygen that accept electrons prevent rapid kinetics. Abiotic sulfide oxidation kinetics improve when reduced iron and/or manganese are oxidized by oxygen to form oxidized metals which in turn oxidize sulfide. Biological sulfur oxidation relies on enzymes that have evolved to overcome these kinetic constraints to affect rapid sulfide oxidation. Here we review the available thermodynamic and kinetic data for H(2)S and HS⢠as well as O(2), reactive oxygen species, nitrate, nitrite, and NO(x) species. We also present new kinetic data for abiotic sulfide oxidation with oxygen in trace metal clean solutions that constrain abiotic rates of sulfide oxidation in metal free solution and agree with the kinetic and thermodynamic calculations. Moreover, we present experimental data that give insight on rates of chemolithotrophic and photolithotrophic sulfide oxidation in the environment. We demonstrate that both anaerobic photolithotrophic and aerobic chemolithotrophic sulfide oxidation rates are three or more orders of magnitude higher than abiotic rates suggesting that in most environments biotic sulfide oxidation rates will far exceed abiotic rates due to the thermodynamic and kinetic constraints discussed in the first section of the paper. Such data reshape our thinking about the biotic and abiotic contributions to sulfide oxidation in the environment.