RESUMEN
Siderophores are low molecular weight iron-chelating molecules that many organisms secrete to scavenge ferric iron from the environment. While cyanobacteria inhabit a wide range of environments with poor iron availability, only two siderophore families have been characterized from this phylum. Herein, we sought to investigate siderophore production in the marine genus, Leptolyngbya. A 12 open reading frame (14.5 kb) putative nonribosomal peptide synthetase-independent siderophore biosynthesis gene cluster, identified in the genome of Leptolyngbya sp. PCC 7376, was cloned and heterologously expressed in Escherichia coli. Under iron-limiting conditions, expression strains harboring the first seven genes (lidA to lidF), produced a potent siderophore, which was subsequently identified via UPLC-MS/MS and NMR as schizokinen. The enzymes encoded by the remaining genes (lidG1 to lidG5) did not appear to be active in E. coli, therefore their function could not be determined. Bioinformatic analysis revealed gene clusters with high homology to lidA to lidF in phylogenetically and biogeographically diverse cyanobacteria, suggesting that schizokinen-based siderophore production is widespread in this phylum. Siderophore yields in E. coli expression strains were significantly higher than those achieved by Leptolyngbya, highlighting the potential of this platform for producing siderophores of industrial value. IMPORTANCE Iron availability limits the growth of many microorganisms, particularly those residing in high nutrient-low chlorophyll aquatic environments. Therefore, characterizing iron acquisition pathways in phytoplankton is essential for understanding nutrient cycling in our oceans. The results of this study suggest that Leptolyngbya sp. PCC 7376, and many other cyanobacteria, use schizokinen-based iron chelators (siderophores) to scavenge iron from the environment. We have shown that these pathways are amenable to heterologous expression in E. coli, which expands the limited arsenal of known cyanobacterial siderophores and is advantageous for the downstream overproduction of relevant siderophores of ecological and industrial value.
Asunto(s)
Cianobacterias , Sideróforos , Cromatografía Liquida , Cianobacterias/genética , Cianobacterias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Ácidos Hidroxámicos , Hierro/metabolismo , Sideróforos/metabolismo , Espectrometría de Masas en TándemRESUMEN
Paralytic shellfish toxins (PSTs) are neurotoxic alkaloids produced by freshwater cyanobacteria and marine dinoflagellates. Due to their antagonism of voltage-gated sodium channels in excitable cells, certain analogues are of significant pharmacological interest. The biosynthesis of the parent compound, saxitoxin, is initiated with the formation of 4-amino-3-oxo-guanidinoheptane (ethyl ketone) by an unusual polyketide synthase-like enzyme, SxtA. We have heterologously expressed SxtA from Raphidiopsis raciborskii T3 in Escherichia coli and analysed its activity inâ vivo. Ethyl ketone and a truncated analogue, methyl ketone, were detected by HPLC-ESI-HRMS analysis, thus suggesting that SxtA has relaxed substrate specificity inâ vivo. The chemical structures of these products were further verified by tandem mass spectrometry and labelled-precursor feeding with [guanidino-15 N2 ] arginine and [1,2-13 C2 ] acetate. These results indicate that the reactions catalysed by SxtA could give rise to multiple PST variants, including analogues of ecological and pharmacological significance.
Asunto(s)
Cylindrospermopsis/metabolismo , Escherichia coli/metabolismo , Venenos/metabolismo , Saxitoxina/metabolismo , Canales de Sodio Activados por Voltaje/química , Cylindrospermopsis/genética , Escherichia coli/genética , Saxitoxina/genética , Especificidad por SustratoRESUMEN
BACKGROUND: Dolichospermum circinale is a filamentous bloom-forming cyanobacterium responsible for biosynthesis of the paralytic shellfish toxins (PST), including saxitoxin. PSTs are neurotoxins and in their purified form are important analytical standards for monitoring the quality of water and seafood and biomedical research tools for studying neuronal sodium channels. More recently, PSTs have been recognised for their utility as local anaesthetics. Characterisation of the transcriptional elements within the saxitoxin (sxt) biosynthetic gene cluster (BGC) is a first step towards accessing these molecules for biotechnology. RESULTS: In D. circinale AWQC131C the sxt BGC is transcribed from two bidirectional promoter regions encoding five individual promoters. These promoters were identified experimentally using 5' RACE and their activity assessed via coupling to a lux reporter system in E. coli and Synechocystis sp. PCC 6803. Transcription of the predicted drug/metabolite transporter (DMT) encoded by sxtPER was found to initiate from two promoters, PsxtPER1 and PsxtPER2. In E. coli, strong expression of lux from PsxtP, PsxtD and PsxtPER1 was observed while expression from Porf24 and PsxtPER2 was remarkably weaker. In contrast, heterologous expression in Synechocystis sp. PCC 6803 showed that expression of lux from PsxtP, PsxtPER1, and Porf24 promoters was statistically higher compared to the non-promoter control, while PsxtD showed poor activity under the described conditions. CONCLUSIONS: Both of the heterologous hosts investigated in this study exhibited high expression levels from three of the five sxt promoters. These results indicate that the majority of the native sxt promoters appear active in different heterologous hosts, simplifying initial cloning efforts. Therefore, heterologous expression of the sxt BGC in either E. coli or Synechocystis could be a viable first option for producing PSTs for industrial or biomedical purposes.
Asunto(s)
Proteínas Bacterianas/genética , Cianobacterias/genética , Saxitoxina/biosíntesis , Cianobacterias/metabolismo , Modelos Genéticos , Familia de Multigenes , Regiones Promotoras Genéticas , Saxitoxina/genéticaRESUMEN
The microcystins are a large group of cyclic peptide hepatotoxins produced by several genera of freshwater cyanobacteria. The genes responsible for microcystin biosynthesis are encoded within a large (â¼55 kbp) gene cluster, mcyA-J. The recent establishment of a cyanotoxin heterologous expression system in Escherichia coli has provided the means to study microcystin biosynthesis in a genetically tractable, rapidly growing host. Using this system, we demonstrate that deletion of the ABC-transporter, mcyH, and dehydrogenase, mcyI, abolishes microcystin production, while deletion of the O-methyltransferase, mcyJ, results in the production of the demethylated (DM) toxin [d-Asp3, DMAdda5]microcystin-LR. Both methylated and DM toxin variants were heterologously produced at high titers and efficiently exported into the extracellular medium, enabling easy purification. The results show that the mcy gene cluster can be engineered in E. coli to study the function of its individual components and direct the synthesis of particular microcystin variants. This technology could potentially be applied to other natural products of ecological and biomedical significance.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Proteínas Bacterianas/genética , Microcistinas/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Cromatografía Líquida de Alta Presión , Escherichia coli/metabolismo , Microcistinas/análisis , Microcistinas/química , Familia de Multigenes , Mutagénesis , Proteína O-Metiltransferasa/deficiencia , Proteína O-Metiltransferasa/genética , Espectrometría de Masas en TándemRESUMEN
Covering: up to 2018 Marine and freshwater cyanobacteria produce a variety of toxic compounds that pose a threat to the health of humans, livestock and natural ecosystems world-wide. Significant research efforts have been directed towards understanding the biosynthesis of these cyanotoxins in an attempt to reduce their deleterious effects on water quality and, more recently, to harness their biotechnological potential. While a variety of complementary methods (such as bioinformatic analyses and isotope feeding studies) have been employed over the last three decades to address knowledge gaps in this field, this review focuses on the utility of heterologous expression and biochemical studies, including emerging technologies for engineering and expressing complete cyanotoxin gene clusters.
Asunto(s)
Toxinas Bacterianas/biosíntesis , Cianobacterias/metabolismo , Animales , Vías Biosintéticas , Agua Dulce/microbiología , Humanos , Toxinas Marinas/química , Toxinas Marinas/toxicidad , Agua de Mar/microbiologíaRESUMEN
The neurotoxin saxitoxin and related paralytic shellfish toxins are produced by multiple species of cyanobacteria and dinoflagellates. This study investigates the two saxitoxin-producing strains of Scytonema crispum, CAWBG524 and CAWBG72, isolated in New Zealand. Each strain was previously reported to have a distinct paralytic shellfish toxin profile, a rare observation between strains within the same species. Sequencing of the saxitoxin biosynthetic clusters ( sxt) from S. crispum CAWBG524 and S. crispum CAWBG72 revealed the largest sxt gene clusters described to date. The distinct toxin profiles of each strain were correlated to genetic differences in sxt tailoring enzymes, specifically the open-reading frame disruption of the N-21 sulfotransferase sxtN, adenylylsulfate kinase sxtO, and the C-11 dioxygenase sxtDIOX within S. crispum CAWBG524 via genetic insertions. Heterologous overexpression of SxtN allowed for the proposal of saxitoxin and 3'-phosphoadenosine 5'-phosphosulfate as substrate and cofactor, respectively, using florescence binding assays. Further, catalytic activity of SxtN was confirmed by the in vitro conversion of saxitoxin to the N-21 sulfonated analog gonyautoxin 5, making this the first known report to biochemically confirm the function of a sxt tailoring enzyme. Further, SxtN could not convert neosaxitoxin to its N-21 sulfonated analog gonyautoxin 6, indicating paralytic shellfish toxin biosynthesis most likely occurs along a predefined route. In this study, we identified key steps toward the biosynthetic conversation of saxitoxin to other paralytic shellfish toxins.
Asunto(s)
Familia de Multigenes , Neurotoxinas/clasificación , Neurotoxinas/genética , Saxitoxina/clasificación , Saxitoxina/genética , Cianobacterias/genética , Dioxigenasas/genética , Genes Bacterianos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Neurotoxinas/química , Fosfoadenosina Fosfosulfato/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Filogenia , Unión Proteica , Saxitoxina/análogos & derivados , Saxitoxina/síntesis química , Saxitoxina/química , Sulfotransferasas/química , Sulfotransferasas/genética , Sulfotransferasas/metabolismo , Transposasas/genéticaRESUMEN
Cylindrospermopsin, a cytotoxin from cyanobacteria, is biosynthesized by a complex pathway, which involves CyrI, an iron and 2-oxoglutarate dependent hydroxylase that transforms 7-deoxy-cylindrospermopsin into cylindrospermopsin and its epimer, 7-epi-cylindrospermopsin, in the last step. The activity of CyrI from Oscillatoria sp. PCC 7926 depends on Fe(II) (Kmâ¯=â¯2.1⯵M), and 2-oxoglutarate (Kmâ¯=â¯3.2⯵M), and is strongly inhibited by 7-deoxy-cylindrospermopsin at concentration higher than 1⯵M. Using tryptophan fluorescence, we measured the binding to CyrI of Fe(II) (KDâ¯=â¯0.02⯵M) and 2-oxoglutarate (KDâ¯=â¯53⯵M and KDâ¯=â¯1.1⯵M in the absence or presence of 10⯵M Fe(II), respectively). The Oscillatoria sp. PCC 6506 CyrI mutants H157A, D159A, H247A, and R257A were all inactive, and impaired in the binding of Fe(II) or 2-oxoglutarate, confirming the identity of the iron ligands and the role of R257 in the binding of 2-oxoglutarate. We constructed several chimeric enzymes using the Oscillatoria sp. PCC 7926 CyrI protein (stereoselective) and that from Oscillatoria sp. PCC 6506 (not stereoselective) to help understanding the structural factors that influence the stereoselectivity of the hydroxylation. Our data suggest that a predicted α-helix in CyrI (positions 87-108) seems to modulate the stereoselectivity of the reaction.
Asunto(s)
Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Vías Biosintéticas , Oxigenasas de Función Mixta/metabolismo , Oscillatoria/metabolismo , Uracilo/análogos & derivados , Alcaloides , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Sitios de Unión , Toxinas de Cianobacterias , Hidroxilación , Hierro/metabolismo , Ácidos Cetoglutáricos/metabolismo , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/genética , Mutagénesis Sitio-Dirigida , Oscillatoria/química , Oscillatoria/genética , Alineación de Secuencia , Estereoisomerismo , Uracilo/química , Uracilo/metabolismoRESUMEN
Phosphopantetheinyl transferases catalyze the post-translational modification of carrier proteins involved in both primary and secondary metabolism. The functional expression of polyketide synthases and nonribosomal peptide synthetases requires the activation of all carrier protein domains by phosphopantetheinyl transferases. Here we describe the characterization of five bacterial phosphopantetheinyl transferases by their substrate specificity and catalytic efficiency of four cyanobacterial carrier proteins. Comparative in vitro phosphopantetheinylation analysis showed Sfp possesses the highest catalytic efficiency over various carrier proteins. In vivo coexpression of phosphopantetheinyl transferases with carrier proteins revealed a broad range substrate specificity of phosphopantetheinyl transferases; all studied phosphopantetheinyl transferases were capable of converting apo- carrier proteins, sourced from diverse biosynthetic enzymes, to their active holo form. Phosphopantetheinyl transferase coexpression with the hybrid nonribosomal peptide synthetases/polyketide synthases responsible for microcystin biosynthesis confirmed that the higher in vitro activity of Sfp translated in vivo to a higher yield of production.
Asunto(s)
Proteínas Bacterianas/metabolismo , Productos Biológicos/metabolismo , Proteínas Portadoras/metabolismo , Cianobacterias/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Simulación por Computador , Cianobacterias/química , Enlace de Hidrógeno , Microcistinas/genética , Microcistinas/metabolismo , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Sintasas Poliquetidas/metabolismo , Procesamiento Proteico-Postraduccional , Especificidad por Sustrato , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genéticaRESUMEN
The cyanobacterium Dolichospermum circinale (formerly Anabaena circinalis) is responsible for neurotoxic saxitoxin-producing blooms in Australia. Previous studies have reported distinct isolates of toxic D. circinale producing different saxitoxin analogues at varying amounts, but the mechanisms responsible remain poorly understood. To assess the characteristics that may be responsible for this variance, a morphological, molecular and chemical survey of 28 Anabaena isolates was conducted. Morphological characteristics, presence or absence of saxitoxin biosynthetic genes and toxin amount and profile were assessed. The 28 isolates were collected from 16 locations. A correlation between the size of the isolates and its reported toxicity or geographical location could not be found. Molecular screening for the presence of several sxt genes revealed eight out of the 28 strains harboured the sxt gene cluster and all tailoring genes except sxtX. Furthermore, the presence of PSTs was correlated with the presence of the sxt cluster using quantitative pre-column oxidation high performance liquid chromatography with fluorescence detection (HPLC-FLD) and LC-MS/MS. Interestingly, isolates differed in the amount and type of toxins produced, with the eight toxic strains containing the core and tailoring biosynthetic genes while non-toxic strains were devoid of these genes. Moreover, the presence of sxt tailoring genes in toxic strains correlated with the biosynthesis of analogues. A greater understanding of toxin profile/quantity from distinct sites around Australia will aid the management of these at-risk areas and provide information on the molecular control or physiological characteristics responsible for toxin production.
Asunto(s)
Cianobacterias/genética , Saxitoxina/genética , Australia , Cianobacterias/clasificación , Cianobacterias/citología , ADN Bacteriano , Toxinas Marinas/biosíntesis , Toxinas Marinas/genética , Familia de Multigenes , ARN Ribosómico 16S , Saxitoxina/análogos & derivados , Saxitoxina/biosíntesis , Análisis de Secuencia de ADN , Intoxicación por MariscosRESUMEN
Microcystins are globally the most commonly occurring freshwater cyanotoxins, causing acute poisoning and chronically inducing hepatocellular carcinoma. However, the detection and toxicological study of microcystins is hampered by the limited availability and high cost of pure toxin standards. Biosynthesis of microcystin variants in a fast-growing heterologous host offers a promising method of achieving reliable and economically viable alternative to isolating toxin from slow-growing cyanobacterial cultures. Here, we report the heterologous expression of recombinant microcystin synthetases in Escherichia coli to produce [d-Asp3]microcystin-LR and microcystin-LR. We assembled a 55 kb hybrid polyketide synthase/nonribosomal peptide synthetase gene cluster from Microcystis aeruginosa PCC 7806 using Red/ET recombineering and replaced the native promoters with an inducible PtetO promoter to yield microcystin titers superior to M. aeruginosa. The expression platform described herein can be tailored to heterologously produce a wide variety of microcystin variants, and potentially other cyanobacterial natural products of commercial relevance.
Asunto(s)
Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/genética , Cianobacterias/genética , Escherichia coli/genética , Microbiología Industrial/métodos , Toxinas Marinas/biosíntesis , Toxinas Marinas/genética , Microcistinas/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Cianobacterias/enzimología , Toxinas de Cianobacterias , Toxinas Marinas/metabolismo , Microcistinas/biosíntesis , Microcistinas/genética , Microcistinas/metabolismo , Familia de Multigenes/genética , Péptido Sintasas/genética , Regiones Promotoras Genéticas/genéticaRESUMEN
Mycosporine-like amino acids (MAAs) are an important class of secondary metabolites known for their protection against UV radiation and other stress factors. Cyanobacteria produce a variety of MAAs, including shinorine, the active ingredient in many sunscreen creams. Bioinformatic analysis of the genome of the soil-dwelling cyanobacterium Cylindrospermum stagnale PCC 7417 revealed a new gene cluster with homology to MAA synthase from Nostoc punctiforme This newly identified gene cluster is unusual because it has five biosynthesis genes (mylA to mylE), compared to the four found in other MAA gene clusters. Heterologous expression of mylA to mylE in Escherichia coli resulted in the production of mycosporine-lysine and the novel compound mycosporine-ornithine. To our knowledge, this is the first time these compounds have been heterologously produced in E. coli and structurally characterized via direct spectral guidance. This study offers insight into the diversity, biosynthesis, and structure of cyanobacterial MAAs and highlights their amenability to heterologous production methods. IMPORTANCE: Mycosporine-like amino acids (MAAs) are significant from an environmental microbiological perspective as they offer microbes protection against a variety of stress factors, including UV radiation. The heterologous expression of MAAs in E. coli is also significant from a biotechnological perspective as MAAs are the active ingredient in next-generation sunscreens.
Asunto(s)
Aminoácidos/biosíntesis , Cianobacterias/metabolismo , Ciclohexanoles/metabolismo , Escherichia coli/metabolismo , Lisina/biosíntesis , Ornitina/biosíntesis , Aminoácidos/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cianobacterias/genética , Ciclohexanoles/química , Escherichia coli/genética , Lisina/química , Ornitina/químicaRESUMEN
UNLABELLED: The mycosporine-like amino acids (MAAs) are a group of small molecules with a diverse ecological distribution among microorganisms. MAAs have a range of physiological functions, including protection against UV radiation, making them important from a biotechnological perspective. In the present study, we identified a putative MAA (mys) gene cluster in two New Zealand isolates of Scytonema cf. crispum (UCFS10 and UCFS15). Homology to "Anabaena-type" mys clusters suggested that this cluster was likely to be involved in shinorine biosynthesis. Surprisingly, high-performance liquid chromatography analysis of S cf. crispum cell extracts revealed a complex MAA profile, including shinorine, palythine-serine, and their hexose-bound variants. It was hypothesized that a short-chain dehydrogenase (UCFS15_00405) encoded by a gene adjacent to the S cf. crispum mys cluster was responsible for the conversion of shinorine to palythine-serine. Heterologous expression of MysABCE and UCFS15_00405 in Escherichia coli resulted in the exclusive production of the parent compound shinorine. Taken together, these results suggest that shinorine biosynthesis in S cf. crispum proceeds via an Anabaena-type mechanism and that the genes responsible for the production of other MAA analogues, including palythine-serine and glycosylated analogues, may be located elsewhere in the genome. IMPORTANCE: Recently, New Zealand isolates of S cf. crispum were linked to the production of paralytic shellfish toxins for the first time, but no other natural products from this species have been reported. Thus, the species was screened for important natural product biosynthesis. The mycosporine-like amino acids (MAAs) are among the strongest absorbers of UV radiation produced in nature. The identification of novel MAAs is important from a biotechnology perspective, as these molecules are able to be utilized as sunscreens. This study has identified two novel MAAs that have provided several new avenues of future research related to MAA genetics and biosynthesis. Further, we have revealed that the genetic basis of MAA biosynthesis may not be clustered on the genome. The identification of the genes responsible for MAA biosynthesis is vital for future genetic engineering.
Asunto(s)
Aminoácidos/metabolismo , Cianobacterias/genética , Ciclohexanoles/metabolismo , Ciclohexilaminas/metabolismo , Genes Bacterianos , Glicina/análogos & derivados , Familia de Multigenes , Cianobacterias/metabolismo , Glicina/metabolismo , Nueva Zelanda , Análisis de Secuencia de ADN , Protectores Solares/análisisRESUMEN
The production of toxic metabolites by cyanobacterial blooms represents a significant threat to the health of humans and ecosystems worldwide. Here we summarize the current state of the knowledge regarding the genetics, biosynthesis and regulation of well-characterized cyanotoxins, including the microcystins, nodularin, cylindrospermopsin, saxitoxins and anatoxins, as well as the lesser-known marine toxins (e.g. lyngbyatoxin, aplysiatoxin, jamaicamides, barbamide, curacin, hectochlorin and apratoxins).
Asunto(s)
Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/genética , Cianobacterias/fisiología , Toxinas Bacterianas/metabolismo , Cianobacterias/genética , Ecosistema , Regulación Bacteriana de la Expresión Génica , HumanosRESUMEN
We have synthesized cis- and trans-dihydroanatoxin-a and cis- and trans-dihydrohomoanatoxin-a using a short synthetic route. The relative configuration of N-tert-butoxycarbonyl-cis-dihydroanatoxin-a was determined by X-ray crystallography, while that of N-tert-butoxycarbonyl-trans-dihydroanatoxin-a was confirmed by epimerization leading to the cis-diastereoisomer. The relative configuration of N-tert-butoxycarbonyl-trans- and cis-dihydrohomoanatoxin-a was inferred from their NMR spectra. Using an optimized LC-MS/MS analytical method and pure standards we have simultaneously determined anatoxin-a, homoanatoxin-a and their dihydroderivatives in axenic strains of cyanobacteria and in environmental samples from the Tarn River, France. However, in these analytical conditions, the cis- and trans-dihydroanatoxin-a and cis- and trans-dihydrohomoanatoxin-a could not be separated. In axenic strains, the dihydroderivatives represented less than 3% of the total toxin content, while in field samples dihydroanatoxin-a represented from 17% to 90% of the total toxin content. Thus, the reduction of anatoxin-a to dihydroanatoxin-a is predominant in the environment. The ratio of anatoxin-a concentration over that of homoanatoxin-a in axenic strains was variable, and among the eight strains studied we found three exclusive anatoxin-a producers and five producers of homoanatoxin-a and anatoxin-a, the latter representing from 0.5% to 2.0% of the total. In the strains studied, we have amplified by PCR, and sequenced the region of anaG coding for the methylation domain proposed to be responsible for the formation of homoanatoxin-a. The sequences showed at least 88% identity and we could not relate the toxin profile of the strains to the sequence of the methylation domain.
Asunto(s)
Cromatografía Liquida/métodos , Cianobacterias/química , Espectrometría de Masas en Tándem/métodos , Tropanos/análisis , Microbiología del Agua , Cristalografía por Rayos X , Espectroscopía de Resonancia MagnéticaRESUMEN
BACKGROUND: Protein folding in the envelope is a crucial limiting step of protein export and secretion. In order to better understand this process in Lactococcus lactis, a lactic acid bacterium, genes encoding putative exported folding factors like Peptidyl Prolyl Isomerases (PPIases) were searched for in lactococcal genomes. RESULTS: In L. lactis, a new putative membrane PPIase of the cyclophilin subfamily, PpiA, was identified and characterized. ppiA gene was found to be constitutively expressed under normal and stress (heat shock, H(2)O(2)) conditions. Under normal conditions, PpiA protein was synthesized and released from intact cells by an exogenously added protease, showing that it was exposed at the cell surface. No obvious phenotype could be associated to a ppiA mutant strain under several laboratory conditions including stress conditions, except a very low sensitivity to H(2)O(2). Induction of a ppiA copy provided in trans had no effect i) on the thermosensitivity of an mutant strain deficient for the lactococcal surface protease HtrA and ii) on the secretion and stability on four exported proteins (a highly degraded hybrid protein and three heterologous secreted proteins) in an otherwise wild-type strain background. However, a recombinant soluble form of PpiA that had been produced and secreted in L. lactis and purified from a culture supernatant displayed both PPIase and chaperone activities. CONCLUSIONS: Although L. lactis PpiA, a protein produced and exposed at the cell surface under normal conditions, displayed a very moderate role in vivo, it was found, as a recombinant soluble form, to be endowed with folding activities in vitro.
Asunto(s)
Proteínas Bacterianas/metabolismo , Membrana Celular/enzimología , Lactococcus lactis/enzimología , Proteínas de la Membrana/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Pliegue de Proteína , Proteínas Bacterianas/genética , Membrana Celular/genética , Ciclofilinas/genética , Ciclofilinas/metabolismo , Peróxido de Hidrógeno/farmacología , Lactococcus lactis/genética , Proteínas de la Membrana/genética , Oxidantes/farmacología , Isomerasa de Peptidilprolil/genética , Estrés Fisiológico/efectos de los fármacosAsunto(s)
Proteínas Bacterianas/metabolismo , Cianobacterias/enzimología , Oxigenasas/metabolismo , Uracilo/análogos & derivados , Alcaloides , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Toxinas Bacterianas , Biocatálisis , Toxinas de Cianobacterias , Citotoxinas/biosíntesis , Citotoxinas/química , Hidroxilación , Ácidos Cetoglutáricos/química , Cinética , Datos de Secuencia Molecular , Oxigenasas/química , Oxigenasas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Estereoisomerismo , Uracilo/biosíntesis , Uracilo/químicaRESUMEN
We report a draft sequence of the genome of Oscillatoria sp. PCC 6506, a cyanobacterium that produces anatoxin-a and homoanatoxin-a, two neurotoxins, and cylindrospermopsin, a cytotoxin. Beside the clusters of genes responsible for the biosynthesis of these toxins, we have found other clusters of genes likely involved in the biosynthesis of not-yet-identified secondary metabolites.
Asunto(s)
Genoma Bacteriano/genética , Familia de Multigenes/genética , Oscillatoria/genética , Toxinas Bacterianas/genética , Datos de Secuencia Molecular , Familia de Multigenes/fisiologíaRESUMEN
BACKGROUND: Theoretical and experimental data support the geographic differentiation strategy as a valuable tool for detecting loci under selection. In the context of Plasmodium falciparum malaria, few populations have been studied, with limited genomic coverage. METHODS: We examined geographic differentiation in P. falciparum populations on the basis of 12 single-nucleotide polymorphisms (SNPs) in 4 genes encoding drug resistance determinants, 5 SNPs in 2 genes encoding antigens, and a set of 17 putatively neutral SNPs dispersed on 13 chromosomes. We sampled 326 parasite isolates representing 7 P. falciparum populations from regions with varied levels of malaria transmission (Gabon, Kenya, Madagascar, Mali, Mayotte, Haiti, and the Philippines). RESULTS: Frequencies of drug resistance alleles varied considerably among populations (mean F(ST), 0.52). In contrast, allele frequencies varied significantly less for antigenic and neutral SNPs (mean F(ST), 0.16 and 0.24, respectively). This contrasting pattern was more pronounced when only the African populations were considered. Signature of selection was detected for most of the resistant SNPs but not for the antigenic SNPs. CONCLUSION: These data further validate the utility of geographic differentiation for identifying loci under strong positive selection, such as drug resistance loci. This study also provides frequencies of molecular makers of resistance in some overlooked populations.
Asunto(s)
Resistencia a Medicamentos , Malaria Falciparum/parasitología , Plasmodium falciparum/clasificación , Plasmodium falciparum/genética , Polimorfismo de Nucleótido Simple , Adaptación Biológica , Adolescente , Adulto , África , Anciano , Anciano de 80 o más Años , Niño , Preescolar , ADN Protozoario/química , ADN Protozoario/genética , Femenino , Genes Protozoarios , Geografía , Haití , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Filipinas , Plasmodium falciparum/aislamiento & purificación , Adulto JovenRESUMEN
Cylindrospermopsin is a cytotoxin produced by Cylindrospermopsis raciborskii and other cyanobacteria that has been implicated in human intoxications. We report here the complete sequence of the gene cluster responsible for the biosynthesis of this toxin in Oscillatoria sp. strain PCC 6506. This cluster of genes was found to be homologous with that of C. raciborskii but with a different gene organization. Using an enzyme-linked immunosorbent assay and an optimized liquid chromatography analytical method coupled to tandem mass spectrometry, we detected 7-epicylindrospermopsin, cylindrospermopsin, and 7-deoxycylindrospermopsin in the culture medium of axenic Oscillatoria PCC 6506 at the following relative concentrations: 68.6%, 30.2%, and 1.2%, respectively. We measured the intracellular and extracellular concentrations, per mg of dried cells of Oscillatoria PCC 6506, of 7-epicylindrospermopsin (0.18 microg/mg and 0.29 microg/mg, respectively) and cylindrospermopsin (0.10 microg/mg and 0.11 microg/mg, respectively). We showed that these two toxins accumulated in the culture medium of Oscillatoria PCC 6506 but that the ratio (2.5 +/- 0.3) was constant with 7-epicylindrospermopsin being the major metabolite. We also determined the concentrations of these toxins in culture media of other Oscillatoria strains, PCC 6407, PCC 6602, PCC 7926, and PCC 10702, and found that, except for PCC 6602, they all produced 7-epicylindrospermopsin and cylindrospermopsin, with the former being the major toxin, except for PCC 7926, which produced very little 7-epicylindrospermopsin. All the cylindrospermopsin producers studied gave a PCR product using specific primers for the amplification of the cyrJ gene from genomic DNA.