RESUMO
The origin and evolution of photosynthesis have long remained enigmatic due to a lack of sequence information of photosynthesis genes across the entire photosynthetic domain. To probe early evolutionary history of photosynthesis, we obtained new sequence information of a number of photosynthesis genes from the green sulfur bacterium Chlorobium tepidum and the green nonsulfur bacterium Chloroflexus aurantiacus. A total of 31 open reading frames that encode enzymes involved in bacteriochlorophyll/porphyrin biosynthesis, carotenoid biosynthesis, and photosynthetic electron transfer were identified in about 100 kilobase pairs of genomic sequence. Phylogenetic analyses of multiple magnesium-tetrapyrrole biosynthesis genes using a combination of distance, maximum parsimony, and maximum likelihood methods indicate that heliobacteria are closest to the last common ancestor of all oxygenic photosynthetic lineages and that green sulfur bacteria and green nonsulfur bacteria are each other's closest relatives. Parsimony and distance analyses further identify purple bacteria as the earliest emerging photosynthetic lineage. These results challenge previous conclusions based on 16S ribosomal RNA and Hsp60/Hsp70 analyses that green nonsulfur bacteria or heliobacteria are the earliest phototrophs. The overall consensus of our phylogenetic analysis, that bacteriochlorophyll biosynthesis evolved before chlorophyll biosynthesis, also argues against the long-held Granick hypothesis.
Assuntos
Bactérias/genética , Chlorobi/genética , Chlorobi/metabolismo , Evolução Molecular , Fotossíntese/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Bacterioclorofilas/biossíntese , Bacterioclorofilas/genética , Clorofila/biossíntese , Clorofila A , Cianobactérias/genética , Cianobactérias/metabolismo , Genes Bacterianos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
A phytochrome-like protein called Ppr was discovered in the purple photosynthetic bacterium Rhodospirillum centenum. Ppr has a photoactive yellow protein (PYP) amino-terminal domain, a central domain with similarity to phytochrome, and a carboxyl-terminal histidine kinase domain. Reconstitution experiments demonstrate that Ppr covalently attaches the blue light-absorbing chromophore p-hydroxycinnamic acid and that it has a photocycle that is spectrally similar to, but kinetically slower than, that of PYP. Ppr also regulates chalcone synthase gene expression in response to blue light with autophosphorylation inhibited in vitro by blue light. Phylogenetic analysis demonstrates that R. centenum Ppr may be ancestral to cyanobacterial and plant phytochromes.
Assuntos
Proteínas de Bactérias/química , Fotorreceptores Microbianos , Fitocromo/química , Rhodospirillum/química , Aciltransferases/genética , Sequência de Aminoácidos , Apoproteínas/química , Apoproteínas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Quimiotaxia , Clonagem Molecular , Ácidos Cumáricos/metabolismo , Regulação Bacteriana da Expressão Gênica , Histidina Quinase , Luz , Dados de Sequência Molecular , Mutação , Fosforilação , Filogenia , Propionatos , Proteínas Quinases/metabolismo , Rhodospirillum/genética , Rhodospirillum/fisiologia , Alinhamento de SequênciaRESUMO
Bacteriophage lambda integration and excision occur by reciprocal recombination within a 15-base homologous core region present in the recombining attachment (att) sites. Strand exchange within the core occurs at precise nucleotide positions, which define an overlap region in which the products of recombination contain DNA strands derived from different parents. In order to define the role of sequence homology during recombination we have constructed point mutations within the core and assayed their effects in vivo and in vitro on site-specific recombination. Two of the mutations are located at position -3 of the core, which is one base-pair outside of the overlap region where strand exchange occurs. These mutations do not affect integrative or excisive recombination, thereby suggesting that homology outside the overlap region is not required for recombination. Two other mutations are located at position -2 of the core, which is one base-pair within the overlap region. These mutations show severely depressed integrative and excisive recombination activities in vitro and in vivo when recombined against wild-type att sites. However, the -2 mutations show normal recombination activity when recombined against att sites containing the homologous mutation, thereby suggesting that homology-dependent DNA interactions are required within the overlap region for effective recombination. In vitro recombination between homoduplex attP sites and heteroduplex attB sites demonstrated that the DNA interactions require only one strand of the attB overlap region to be homologous to attP in order to promote recombination.
Assuntos
Bacteriófago lambda/genética , DNA Viral/genética , Recombinação Genética , Sítios de Ligação Microbiológicos , Sequência de Bases , Eletroforese em Gel de Poliacrilamida , Lisogenia , Mutação , Ácidos Nucleicos Heteroduplexes/genéticaRESUMO
The cbb(I) and cbb(II) operons encode structural genes which are important for carbon dioxide fixation via the Calvin-Benson-Bassham reductive pentose phosphate pathway in Rhodobacter capsulatus. Each operon is regulated by cognate LysR-type transcriptional activators, CbbR(I) and CbbR(II), with the product of the cbbR(I) gene, CbbR(I), able to control its own transcription under some growth conditions. Furthermore, CbbR(I) may at least partially regulate the cbb(II) operon, with significant, yet regulated transcription of the cbb(II) operon occurring in the absence of any CbbR. These results suggested the importance of additional regulators. Thus, in addition to the rather specific control exerted by CbbR, a more globally significant regulatory system, the RegA-RegB (PrrA-PrrB) two-component system, was found to contribute to transcriptional regulation of each cbb operon. The regA and regB mutant strains were found to contain constitutive levels of form I and form II RubisCO, the major proteins encoded by the cbb(I) and cbb(II) operons, respectively. In addition, DNaseI footprint analyses indicated that RegA*, a constitutively active mutant form of RegA, binds specifically to cbb(I) and cbb(II) promoter-operator regions. CbbR(I), CbbR(II), and RegA binding loci were localized relative to transcription start sites, leading to a coherent picture of how each of these regulators interacts with specific promoter-operator sequences of the cbb operons.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Regulon/genética , Rhodobacter capsulatus/genética , Transativadores , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Pegada de DNA , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I , Histidina Quinase , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Regiões Operadoras Genéticas/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rhodobacter capsulatus/enzimologia , Rhodobacter capsulatus/crescimento & desenvolvimento , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/genética , Transcrição Gênica/genéticaRESUMO
Mutations that map in or delete the attenuator of the threonine (thr) operon of Escherichia coli were isolated and characterized. These mutations disrupt or delete the transcription termination structure encoded by the attenuator leading to increased transcriptional readthrough into the thr operon structural genes. Most of the base substitutions and single base-pair insertions and deletions map in the G + C-rich region of dyad symmetry in the attenuator and decrease the calculated stabilities of the attenuator RNA secondary structures to similar extents (from -30.8 kcal/mol to approximately -21 kcal/mol). Most of the mutants showed a three- to fourfold increase in homoserine dehydrogenase (thrA gene product) synthesis relative to the wild-type parent strain. The mutation in one mutant (thrL153 + G) lowered the calculated stability of the RNA secondary structure only slightly (from -30.8 to 27.8 kcal/mol) but the mutant still exhibited high levels of homoserine dehydrogenase synthesis. In addition, three base substitution mutants (thrL135U, thrL139A and thrL156U) showed only slightly (1.5 to 2-fold) elevated levels of homoserine dehydrogenase activity, even though the calculated stabilities of the attenuator RNA secondary structures were reduced as much as most of the other mutants. Two of the mutations (thrL135U and thrL156U) mapped in the G + C-rich-A + T-rich junction of the attenuator. The third mutation (thrL139A) creates an A X C pair in the center of the G + C-rich region of the attenuator stem. The results obtained for these mutants show that the stability of the RNA secondary structure does not always correlate with the efficiency of transcription termination. Finally, analysis of the base changes in the substitution mutations showed that the mutational changes do not appear to be random.
Assuntos
Genes Reguladores , Mutação , Óperon , Regiões Terminadoras Genéticas , Treonina/genética , Transcrição Gênica , Sequência de Bases , DNA Bacteriano/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Genes , Genes Bacterianos , Homosserina Desidrogenase/metabolismo , Temperatura Alta , Lisogenia , Conformação de Ácido Nucleico , RNA Bacteriano/genéticaRESUMO
Integrative recombination between specific attachment (att) regions of the bacteriophage lambda genome (attP) and the Escherichia coli genome (attB) results in a prophage flanked by the hybrid recombinant sites attL and attR. Each att site contains sequences to which proteins involved in recombination bind. Using site-directed mutagenesis, we have constructed a related set of point mutations within each of the five Int "arm-type" binding sites located within attP, attL and attR. Footprint analyses of binding demonstrate that mutating the arm-type sites significantly disrupts the binding of Int. Recombination analyses of mutant att sites in vivo and in vitro demonstrate that only three wild-type arm-type sites within attP are required for efficient integrative recombination. Similar analyses demonstrate that efficient excision can occur with two other different sets of wild-type arm-type sites in attL and attR. These results demonstrate that integrative and excisive recombination may involve interactions of Int with distinct and different subsets of arm-type sites.
Assuntos
Sítios de Ligação Microbiológicos , Bacteriófago lambda/genética , Lisogenia , Mutação , Sítios de Ligação , DNA Nucleotidiltransferases/genética , DNA Viral/análise , Escherichia coli/genética , Integrases , Modelos Genéticos , Recombinação Genética , Proteínas Virais/genéticaRESUMO
Previous studies have established that most if not all of the genes required for synthesis of the Rhodobacter capsulatus essential photosystem are clustered on a 46 kb region of the chromosome known as the photosynthesis gene cluster. This region has recently been sequenced in its entirety by Hearst and co-workers, revealing the existence of 23 open reading frames, many of which are thought to be involved in the synthesis of bacteriochlorophyll. In this study we have undertaken a systematic directed mutational analysis of 12 open reading frames in the photosynthesis gene cluster to evaluate whether individual open reading frames have a role in photopigment biosynthesis. The results of this analysis demonstrate that mutations constructed in seven open reading frames resulted in a loss of bacteriochlorophyll biosynthesis, concomitant with the accumulation of specific intermediates in the Mg-tetrapyrrole biosynthetic pathway. One mutation was observed to result in partial disruption of bacteriochlorophyll biosynthesis, leading to the accumulation of bacteriochlorophyll as well as an intermediate in the biosynthetic pathway. We also observed that disruptions constructed in four open reading frames had no discernible effect on the synthesis of photopigments. The results of this analysis are discussed with regard to our current understanding of the role of each of these open reading frames in the synthesis of the R. capsulatus photosystem.
Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Genes Bacterianos , Família Multigênica , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Rhodobacter capsulatus/metabolismo , Proteínas de Bactérias/análise , Cromatografia Líquida de Alta Pressão , Análise Mutacional de DNA , Complexos de Proteínas Captadores de Luz , Dados de Sequência Molecular , Mutagênese Insercional , Fases de Leitura Aberta/genética , Pigmentos Biológicos/análise , Pirróis/análise , Rhodobacter capsulatus/genética , TetrapirróisRESUMO
Recently, we demonstrated that the RegB/RegA two-component regulatory system from Rhodobacter capsulatus functions as a global regulator of metabolic processes that either generate or consume reducing equivalents. For example, the RegB/RegA system controls expression of such energy generating processes as photosynthesis and hydrogen utilization. In addition, RegB/RegA also control nitrogen and carbon fixation pathways that utilize reducing equivalents. Here, we use a combination of DNase I protection and plasmid-based reporter expression studies to demonstrate that RegA directly controls synthesis of cytochrome cbb3 and ubiquinol oxidases that function as terminal electron acceptors in a branched respiratory chain. We also demonstrate that RegA controls expression of cytochromes c2, c(y) and the cytochrome bc1 complex that are involved in both photosynthetic and respiratory electron transfer events. These data provide evidence that the RegB/RegA two-component system has a major role in controlling the synthesis of numerous processes that affect reducing equivalents in Rhodobacter capsulatus.
Assuntos
Proteínas de Bactérias/metabolismo , Transporte de Elétrons/genética , Regulação Bacteriana da Expressão Gênica , Fotossíntese/genética , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Proteínas Quinases , Rhodobacter capsulatus/genética , Sequência de Bases , Sítios de Ligação , Grupo dos Citocromos c/biossíntese , Grupo dos Citocromos c/genética , Citocromos c2 , Pegada de DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/biossíntese , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Complexo IV da Cadeia de Transporte de Elétrons/genética , Indução Enzimática , Genes Bacterianos/genética , Genes Reporter/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Quinona Redutases/biossíntese , Quinona Redutases/genética , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Rhodobacter capsulatus/enzimologia , Rhodobacter capsulatus/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/metabolismoRESUMO
Recombination of phage lambda attachment sites occurs by sequential exchange of the DNA strands at two specific locations. The first exchange produces a Holliday structure, and the second resolves it to recombinant products. Heterology for base substitution mutations in the region between the two strand exchange points (the overlap region) reduces recombination; some mutations inhibit the accumulation of Holliday structures, others inhibit their resolution to recombinant products. To see if heterology also alters the location of the strand exchange points, we determined the segregation pattern of three single and one multiple base pair substitution mutations of the overlap region in crosses with wild type sites. The mutations are known to differ in the severity of their recombination defect and in the stage of strand exchange they affect. The three single mutations behaved similarly: each segregated into both products of recombination, and the two products of a single crossover were frequently nonreciprocal in the overlap region. In contrast, the multiple mutation preferentially segregated into one of the two recombinant products, and the two products of a single crossover appeared to be fully reciprocal. The simplest explanation of the segregation pattern of the single mutations is that strand exchanges occur at the normal locations to produce recombinants with mismatched base pairs that are frequently repaired. The segregation pattern of the multiple mutation is consistent with the view that both strand exchanges usually occur to one side of the mutant site. We suggest that the segregation pattern of a particular mutation is determined by which stage of strand exchange it inhibits and by the severity of the inhibition.
Assuntos
Sítios de Ligação Microbiológicos , Bacteriófago lambda/genética , Troca Genética , DNA Viral/genética , Lisogenia , Recombinação Genética , Sequência de Bases , Cruzamentos Genéticos , Genótipo , MutaçãoRESUMO
We characterized the Rhodobacter capsulatus nifHDK promoter by nucleotide sequencing and nuclease S1 analysis of mRNA-protected DNA probes. Comparison of this promoter to nifP and ntrP promoters from other species reveals extensive homology to the canonical nifP consensus sequence. Using lac fusions we have demonstrated that transcription of the nifHDK operon is totally repressed when the growth medium is supplemented with ammonia, becomes fully derepressed in ammonia-free medium, and proceeds at intermediate levels when other nitrogen sources are used. Based on this information, we constructed plasmid expression vectors in which the rates of transcription from cloned DNA fragments are determined by the nitrogen source used in the growth medium.
Assuntos
Vetores Genéticos , Óperon , Regiões Promotoras Genéticas , Rodopseudomonas/genética , Transcrição Gênica , Sequência de Bases , Clonagem Molecular , Meios de Cultura , Regulação da Expressão Gênica , Genes Bacterianos , Dados de Sequência Molecular , Nitrogênio/farmacologia , Fixação de Nitrogênio/genética , PlasmídeosRESUMO
A genetic enrichment procedure for mutations constructed by oligodeoxynucleotide(oligo)-directed mutagenesis of DNA cloned in M13mp vectors is described. The procedure uses an M13 vector that contains the cloned target DNA and amber (am) mutations within the phage genes I and II. This vector cannot replicate in a suppressor-free (sup degrees) bacterial strain. A gapped heteroduplex is formed by annealing portions of a complementary (-)strand containing wild-type copies of genes I and II to the am-containing template (+)strand. The oligo is annealed to the single-stranded (ss) region and the remaining gaps and nicks are repaired enzymatically to form a closed circular heteroduplex structure. By transfecting the DNA into a sup degrees host we promote the propagation of heteroduplexes with the oligo-containing (-)strand since only this construction contains the wild-type copies of genes I and II. This procedure eliminates the need for any physical separation of the covalently closed circular DNA that contains the oligo from the ss template. Using this technique we have constructed 17 point mutations with mutation frequencies ranging from 2-20% for single base changes and from 0.3-9% for multiple base changes. In addition, we found that the mutation frequencies were affected by the state of DNA methylation in the (+) and (-)strands.
Assuntos
Mutação , Oligodesoxirribonucleotídeos/genética , Colífagos/genética , Reparo do DNA , DNA Viral/genética , Vetores GenéticosRESUMO
Redox properties of the photosynthetic gene repressor PpsR and the blue-light photoreceptor/antirepressor AppA from Rhodobacter sphaeroides have been characterized. Redox titrations of PpsR reveal the presence of a two-electron couple, with an E (m) value of -320 mV at pH 7.0, which is likely to arise from the reversible conversion of two cysteine thiols to a disulfide. This E (m) value is very much more negative than the E (m) = -180 mV value measured previously at pH 7.0 for the disulfide/dithiol couple in CrtJ, the homolog for PpsR in the closely related bacterium Rhodobacter capsulatus. AppA, a flavin-containing blue-light receptor that is also involved in the regulation of gene expression in R. sphaeroides, contains multiple cysteines in its C-terminal region, two of which function as a redox-active dithiol/disulfide couple with an E (m) value of -325 mV at pH 7.0 in the dark. Titrations of this dithiol/disulfide couple in illuminated samples of AppA indicate that the E (m) value of this disulfide/dithiol couple is -315 mV at pH 7.0, identical to the value obtained for AppA in the dark within the combined experimental uncertainties of the two measurements. The E (m) values of AppA and PpsR demonstrate that these proteins are thermodynamically capable of electron transfer for their activity as an anti-repressor/repressor in R. sphaeroides.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Flavoproteínas/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/metabolismo , Rhodobacter sphaeroides/metabolismo , Transcrição Gênica , Oxirredução , Rhodobacter sphaeroides/genéticaRESUMO
Three open reading frames in the Rhodobacter capsulatus photosynthesis gene cluster, designated F0, F108, and F1025, were disrupted by site-directed mutagenesis. Mutants bearing insertions in these reading frames were defective in converting protoporphyrin IX to magnesium-protoporphyrin monomethyl ester, protochlorophyllide to chlorophyllide a, and magnesium-protoporphyrin monomethyl ester to protochlorophyllide, respectively. These results demonstrate that the genes examined most likely encode enzyme subunits that catalyze steps common to plant and bacterial tetrapyrrole photopigment biosynthetic pathways. The open reading frames were found to be part of a large 11-kilobase operon that encodes numerous genes involved in early steps of the bacteriochlorophyll a biosynthetic pathway.
Assuntos
Bacterioclorofilas/biossíntese , Clorofila/análogos & derivados , Genes Bacterianos , Rodopseudomonas/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Códon/genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Escherichia coli/genética , Dados de Sequência Molecular , Família Multigênica , Mutação , Fotossíntese , Plasmídeos , Mapeamento por Restrição , Rodopseudomonas/crescimento & desenvolvimento , Rodopseudomonas/metabolismoRESUMO
Biosynthesis of the photochemical apparatus by purple nonsulfur photosynthetic bacteria is known to be inhibited by molecular oxygen and high light intensity. Polypeptides that bind bacteriochlorophyll (BChl) to form the light-harvesting I (LH-I) and reaction-center (RC) complexes are encoded by a single transcriptional unit termed the puf operon. In this investigation we demonstrate that the first structural gene in the puf operon (pufQ) of Rhodobacter capsulatus encodes a protein that is required for BChl biosynthesis and that there exists a linear relationship between the amount of pufQ expression and the level of BChl synthesis. Protein sequence similarity exists between PufQ and the region of RC polypeptides that are known to bind BChl and quinone. These observations suggest that pufQ may regulate BChl biosynthesis by a "carrier polypeptide" mechanism as originally proposed by Lascelles.
Assuntos
Bacterioclorofilas/biossíntese , Clorofila/análogos & derivados , Óperon , Rodopseudomonas/genética , Sequência de Aminoácidos , Sequência de Bases , Regulação da Expressão Gênica , Dados de Sequência Molecular , Transcrição GênicaRESUMO
Most species of photosynthetic bacteria synthesize their photosynthetic apparatus only under conditions of reduced oxygen tension. To a large extent, this phenomenon is dependent upon anaerobic induction of photosynthesis gene expression. Here we report an example of a regulatory gene, regA, that is involved in transactivating anaerobic expression of the photosynthetic apparatus. We show that RegA is itself responsible for differential induction of light-harvesting and reaction center gene expression relative to operons for photopigment biosynthesis. Surprisingly, strains disrupted for regA were found to retain normal photosynthetic growth capabilities under high light intensities. We further show that photosynthetic growth in the absence of transactivating structural gene expression is a consequence of the superoperonal organization of the photosynthetic gene cluster.
Assuntos
Complexo de Proteínas do Centro de Reação Fotossintética/genética , Rhodobacter capsulatus/genética , Transativadores/genética , Sequência de Aminoácidos , Sequência de Bases , Regulação Bacteriana da Expressão Gênica , Biblioteca Genômica , Luz , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Rhodobacter capsulatus regulates synthesis of bacteriochlorophyll a in response to changes in oxygen partial pressure and light intensity. One early model proposed that this regulation involved a carrier polypeptide that functions to tether tetrapyrrole intermediates to the membrane. In the present study we isolated tetrapyrrole intermediates accumulated in three strains of R. capsulatus that contain mutations which block bacteriochlorophyll a biosynthesis at different steps of the magnesium branch of the pathway. Each of the tetrapyrrole intermediates was shown to be associated with the same 32 kDa polypeptide, as indicated by similar electrophoretic mobility and antigenic cross-reactivity with polyclonal antisera. The 32 kDa pigment-associated protein was further found to have an electrophoretic mobility, antigenic cross-reactivity and N-terminal sequence identical with those of the previously characterized major outer-membrane porin protein of R. capsulatus.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Bacterioclorofilas/biossíntese , Canais Iônicos/metabolismo , Pirróis/metabolismo , Rhodobacter capsulatus/metabolismo , Sequência de Aminoácidos , Western Blotting , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Porinas , TetrapirróisRESUMO
Photosynthetic bacteria respond to alterations in light conditions by migrating to locations that allows optimal use of light as an energy source. Studies have indicated that photosynthesis-driven electron transport functions as an attractant signal for motility among purple photosynthetic bacteria. However, it is unclear just how the motility-based signal transduction system monitors electron flow through photosynthesis-driven electron transport. Recently, we have demonstrated that the purple photosynthetic bacterium Rhodospirillum centenum is capable of rapidly moving swarm cell colonies toward infrared light as well as away from visible light. Light-driven colony motility of R. centenum has allowed us to perform genetic dissection of the signaling pathway that affects photosynthesis-driven motility. In this study, we have undertaken sequence and mutational analyses of one of the components of a signal transduction pathway, Ptr, which appears responsible for transmitting a signal from the photosynthesis-driven electron transport chain to the chemotaxis signal transduction cascade. Mutational analysis demonstrates that cells disrupted for ptr are defective in altering motility in response to light, as well as defective in light-dependent release of methanol. We present a model which proposes that Ptr senses the redox state of a component in the photosynthetic cyclic electron transport chain and that Ptr is responsible for transmitting a signal to the chemotaxis machinery to induce a photosynthesis-dependent motility response.
Assuntos
Proteínas de Bactérias , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Rhodospirillum/fisiologia , Sequência de Aminoácidos , Quimiotaxia , Deleção de Genes , Luz , Proteínas de Membrana/química , Metanol/metabolismo , Proteínas Quimiotáticas Aceptoras de Metil , Dados de Sequência Molecular , Fotossíntese , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Rhodospirillum/genética , Análise de Sequência de DNA , Transdução de SinaisRESUMO
The Chlamydomonas reinhardtii chloroplast gene chlL (frxC) is shown to be involved in the light-independent conversion of protochlorophyllide to chlorophyllide. The polypeptide encoded by chlL contains a striking 53% amino acid sequence identity with the bacteriochlorophyll (bch) biosynthesis bchL gene product in the photosynthetic bacterium Rhodobacter capsulatus. In a previous analysis, we demonstrated that bchL was involved in light-independent protochlorophyllide reduction, thereby implicating chlL in light-independent protochlorophyllide reduction in photosynthetic eukaryotes. To perform a functional/mutational analysis of chlL, we utilized particle gun-mediated transformation to disrupt the structural sequence of chlL at its endogenous locus in the chloroplast genome of Chlamydomonas. Transformants for which the multicopy chloroplast genome was homoplasmic for the disrupted chlL allele exhibit a "yellow-in-the-dark" phenotype that we demonstrated to be a result of the dark accumulation of protochlorophyllide. The presence of a chlL homolog in distantly related bacteria and nonflowering land plants, which are thought to be capable of synthesizing chlorophyll in the dark, was also demonstrated by cross-hybridization analysis. In contrast, we observed no cross-hybridization of a probe of chlL to DNA samples from representative angiosperms that require light for chlorophyll synthesis, in support of our conclusion that chlL is involved in light-independent chlorophyll biosynthesis. The role of chlL in protochlorophyllide reduction as well as recent evidence that both light-independent and light-dependent protochlorophyllide reductases may be of bacterial origin are discussed.
Assuntos
Proteínas de Bactérias , Chlamydomonas reinhardtii/genética , Clorofila/biossíntese , Cloroplastos/metabolismo , Complexos de Proteínas Captadores de Luz , Proteínas de Plantas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Chlamydomonas reinhardtii/metabolismo , Clorofila/genética , Cianobactérias/genética , DNA , Luz , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fenótipo , Fotossíntese/genética , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Plantas/genética , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Transformação GenéticaRESUMO
Previous genetic analysis indicated that the photosynthesis gene cluster from Rhodobacter capsulatus coded for the transcription factor, CrtJ, that is responsible for aerobic repression of bacteriochlorophyll, carotenoid, and light harvesting-II gene expression. In this study, we have heterologously overexpressed and purified CrtJ to homogeneity and shown by gel mobility shift assays that CrtJ is biologically active. DNase I footprint analysis confirms molecular genetic studies by showing that CrtJ binds to conserved palindromic sequences that overlap the -10 and -35 promoter regions of the bchC operon. Graphs of the percentage of DNA bound versus protein concentration show sigmoidal curves, which is highly indicative of cooperative binding of CrtJ to the two palindromic sites. A binding constant for interaction of CrtJ with the palindrome that spans the -10 region was calculated to be 4.8 x 10(-9) M, whereas affinity for the palindrome that spans the -35 region was found to be 2.9 x 10(-9) M. Binding of CrtJ to the bchC promoter region was also found to be redox-sensitive, with CrtJ exhibiting a 4.5-fold higher binding affinity under oxidizing versus reducing conditions.
Assuntos
Proteínas de Bactérias , Bacterioclorofilas/biossíntese , Carotenoides/biossíntese , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Complexo de Proteínas do Centro de Reação Fotossintética/biossíntese , Rhodobacter capsulatus/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação , Sequência Conservada , Pegada de DNA , Proteínas de Ligação a DNA/biossíntese , Desoxirribonuclease I , Genes Bacterianos , Complexos de Proteínas Captadores de Luz , Dados de Sequência Molecular , Família Multigênica , Oxirredução , Oxirredutases/biossíntese , Oxirredutases/genética , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/biossínteseRESUMO
A chemotaxis gene cluster from the photosynthetic bacterium Rhodospirillum centenum has been cloned, sequenced, and analyzed for the control of transcription during swimmer-to-swarm cell differentiation. The first gene of the operon (cheAY) codes for a large 108-kDa polypeptide with an amino-terminal domain that is homologous to CheA and a carboxyl terminus that is homologous to CheY. cheAY is followed by cheW, an additional homolog of cheY, cheB, and cheR. Sequence analysis indicated that all of the che genes are tightly compacted with the same transcriptional polarity, suggesting that they are organized in an operon. Cotranscription of the che genes was confirmed by demonstrating through Western blot analysis that insertion of a polar spectinomycin resistance gene in cheAY results in loss of cheR expression. The promoter for the che operon was mapped by primer extension analysis as well as by the construction of promoter reporter plasmids that include several deletion intervals. This analysis indicated that the R. centenum che operon utilizes two promoters; one exhibits a sigma 70-like sequence motif, and the other exhibits a sigma 54-like motif. Expression of the che operon is shown to be relatively constant for swimmer cells which contain a single flagellum and for swarm cells that contain multiple lateral flagella.