RESUMEN
Microorganisms represent the most abundant biomass on the planet; however, because of several cultivation technique limitations, most of this genetic patrimony has been inaccessible. Due to the advent of metagenomic methodologies, such limitations have been overcome. Prevailing over these limitations enabled the genetic pool of non-cultivable microorganisms to be exploited for improvements in the development of biotechnological products. By utilising a metagenomic approach, we identified a new gene related to biosurfactant production and hydrocarbon degradation. Environmental DNA was extracted from soil samples collected on the banks of the Jundiaí River (Natal, Brazil), and a metagenomic library was constructed. Functional screening identified the clone 3C6, which was positive for the biosurfactant protein and revealed an open reading frame (ORF) with high similarity to sequences encoding a hypothetical protein from species of the family Halobacteriaceae. This protein was purified and exhibited biosurfactant activity. Due to these properties, this protein was named metagenomic biosurfactant protein 1 (MBSP1). In addition, E. coli RosettaTM (DE3) strain cells transformed with the MBSP1 clone showed an increase in aliphatic hydrocarbon degradation. In this study, we described a single gene encoding a protein with marked tensoactive properties that can be produced in a host cell, such as Escherichia coli, without substrate dependence. Furthermore, MBSP1 has been demonstrated as the first protein with these characteristics described in the Archaea or Bacteria domains.
Asunto(s)
Proteínas Bacterianas/metabolismo , Halobacteriaceae/metabolismo , Metabolismo de los Lípidos , Aceites/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Halobacteriaceae/clasificación , Halobacteriaceae/genética , Hidrocarburos/metabolismo , Sistemas de Lectura Abierta , Filogenia , Conformación Proteica , Relación Estructura-Actividad , Tensoactivos/metabolismoRESUMEN
BACKGROUND: Mycoplasma hyopneumoniae, Mycoplasma flocculare and Mycoplasma hyorhinis live in swine respiratory tracts. M. flocculare, a commensal bacterium, is genetically closely related to M. hyopneumoniae, the causative agent of enzootic porcine pneumonia. M. hyorhinis is also pathogenic, causing polyserositis and arthritis. In this work, we present the genome sequences of M. flocculare and M. hyopneumoniae strain 7422, and we compare these genomes with the genomes of other M. hyoponeumoniae strain and to the a M. hyorhinis genome. These analyses were performed to identify possible characteristics that may help to explain the different behaviors of these species in swine respiratory tracts. RESULTS: The overall genome organization of three species was analyzed, revealing that the ORF clusters (OCs) differ considerably and that inversions and rearrangements are common. Although M. flocculare and M. hyopneumoniae display a high degree of similarity with respect to the gene content, only some genomic regions display considerable synteny. Genes encoding proteins that may be involved in host-cell adhesion in M. hyopneumoniae and M. flocculare display differences in genomic structure and organization. Some genes encoding adhesins of the P97 family are absent in M. flocculare and some contain sequence differences or lack of domains that are considered to be important for adhesion to host cells. The phylogenetic relationship of the three species was confirmed by a phylogenomic approach. The set of genes involved in metabolism, especially in the uptake of precursors for nucleic acids synthesis and nucleotide metabolism, display some differences in copy number and the presence/absence in the three species. CONCLUSIONS: The comparative analyses of three mycoplasma species that inhabit the swine respiratory tract facilitated the identification of some characteristics that may be related to their different behaviors. M. hyopneumoniae and M. flocculare display many differences that may help to explain why one species is pathogenic and the other is considered to be commensal. However, it was not possible to identify specific virulence determinant factors that could explain the differences in the pathogenicity of the analyzed species. The M. hyorhinis genome contains differences in some components involved in metabolism and evasion of the host's immune system that may contribute to its growth aggressiveness. Several horizontal gene transfer events were identified. The phylogenomic analysis places M. hyopneumoniae, M. flocculare and M. hyorhinis in the hyopneumoniae clade.
Asunto(s)
Mycoplasma/clasificación , Mycoplasma/genética , Neumonía Porcina por Mycoplasma/microbiología , Sistema Respiratorio/microbiología , Animales , Mapeo Cromosómico , Genoma , Mycoplasma/patogenicidad , Filogenia , Neumonía Porcina por Mycoplasma/genética , Neumonía Porcina por Mycoplasma/patología , Sistema Respiratorio/patología , PorcinosRESUMEN
Glycolytic enzymes, such as fructose-bisphosphate aldolase (FBA) and enolase, have been described as complex multifunctional proteins that may perform non-glycolytic moonlighting functions, but little is known about such functions, especially in parasites. We have carried out in silico genomic searches in order to identify FBA and enolase coding sequences in Echinococcus granulosus, the causative agent of cystic hydatid disease. Four FBA genes and 3 enolase genes were found, and their sequences and exon-intron structures were characterized and compared to those of their orthologs in Echinococcus multilocularis, the causative agent of alveolar hydatid disease. To gather evidence of possible non-glycolytic functions, the expression profile of FBA and enolase isoforms detected in the E. granulosus pathogenic larval form (hydatid cyst) (EgFBA1 and EgEno1) was assessed. Using specific antibodies, EgFBA1 and EgEno1 were detected in protoscolex and germinal layer cells, as expected, but they were also found in the hydatid fluid, which contains parasite's excretory-secretory (ES) products. Besides, both proteins were found in protoscolex tegument and in vitro ES products, further suggesting possible non-glycolytic functions in the host-parasite interface. EgFBA1 modeled 3D structure predicted a F-actin binding site, and the ability of EgFBA1 to bind actin was confirmed experimentally, which was taken as an additional evidence of FBA multifunctionality in E. granulosus. Overall, our results represent the first experimental evidences of alternative functions performed by glycolytic enzymes in E. granulosus and provide relevant information for the understanding of their roles in host-parasite interplay.
Asunto(s)
Echinococcus granulosus/enzimología , Echinococcus granulosus/genética , Fructosa-Bifosfato Aldolasa/genética , Fructosa-Bifosfato Aldolasa/metabolismo , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Fosfopiruvato Hidratasa/genética , Fosfopiruvato Hidratasa/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN de Helmintos/genética , Equinococosis/parasitología , Echinococcus granulosus/patogenicidad , Echinococcus multilocularis/enzimología , Echinococcus multilocularis/genética , Fructosa-Bifosfato Aldolasa/química , Perfilación de la Expresión Génica , Genes de Helminto , Proteínas del Helminto/química , Interacciones Huésped-Parásitos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Fosfopiruvato Hidratasa/química , Mapeo de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Enzootic pneumonia caused by Mycoplasma hyopneumoniae is a major constraint to efficient pork production throughout the world. This pathogen has a small genome with 716 coding sequences, of which 418 are homologous to proteins with known functions. However, almost 42% of the 716 coding sequences are annotated as hypothetical proteins. Alternative methodologies such as threading and comparative modeling can be used to predict structures and functions of such hypothetical proteins. Often, these alternative methods can answer questions about the properties of a model system faster than experiments. In this study, we predicted the structures of seven proteins annotated as hypothetical in M. hyopneumoniae, using the structure-based approaches mentioned above. Three proteins were predicted to be involved in metabolic processes, two proteins in transcription and two proteins where no function could be assigned. However, the modeled structures of the last two proteins suggested experimental designs to identify their functions. Our findings are important in diminishing the gap between the lack of annotation of important metabolic pathways and the great number of hypothetical proteins in the M. hyopneumoniae genome.