RESUMEN
Cell physiology in the weevil Sitophilus oryzae is coordinated by three integrated genomes: nuclear, mitochondrial, and the "S. oryzae principal endosymbiont" (SOPE). SOPE, a cytoplasmic bacterium (2 x 10(3) bacteria per specialized bacteriocyte cell and 3 x 10(6) bacteria per weevil) that belongs to the proteobacteria gamma3-subgroup, is present in all weevils studied. We discovered a fourth prokaryotic genome in somatic and germ tissues of 57% of weevil strains of three species, S. oryzae, Sitophilus zeamais, and Sitophilus granarius, distributed worldwide. We assigned this Gram-negative prokaryote to the Wolbachia group (alpha-proteobacteria), on the basis of 16S rDNA sequence and fluorescence in situ DNA-RNA hybridization (FISH). Both bacteria, SOPE and Wolbachia, were selectively eliminated by combined heat and antibiotic treatments. Study of bacteria involvement in this insect's genetics and physiology revealed that SOPE, which induces the specific differentiation of the bacteriocytes, increases mitochondrial oxidative phosphorylation through the supply of pantothenic acid and riboflavin. Elimination of this gamma3-proteobacterium impairs many physiological traits. By contrast, neither the presence nor the absence of Wolbachia significantly affects the weevil's physiology. Wolbachia, disseminated throughout the body cells, is in particularly high density in the germ cells, where it causes nucleocytoplasmic incompatibility. The coexistence of two distinct types of intracellular proteobacteria at different levels of symbiont integration in insects illustrates the genetic complexity of animal tissue. Furthermore, evolutionary timing can be inferred: first nucleocytoplasm, then mitochondria, then SOPE, and finally Wolbachia. Symbiogenesis, the genetic integration of long-term associated members of different species, in the weevil appears to be a mechanism of speciation (with Wolbachia) and provides a means for animals to acquire new genes that permit better adaptation to the environment (with SOPE).
Asunto(s)
Genoma de Planta , Oryza/genética , Simbiosis/genética , Evolución Biológica , ADN Mitocondrial , Regulación de la Expresión Génica , Genoma Bacteriano , Datos de Secuencia MolecularRESUMEN
The principal intracellular symbiotic bacteria of the cereal weevil Sitophilus oryzae were characterized using the sequence of the 16S rDNA gene (rrs gene) and G + C content analysis. Polymerase chain reaction amplification with universal eubacterial primers of the rrs gene showed a single expected sequence of 1,501 bp. Comparison of this sequence with the available database sequences placed the intracellular bacteria of S. oryzae as members of the Enterobacteriaceae family, closely related to the free-living bacteria, Erwinia herbicola and Escherichia coli, and the endocytobiotic bacteria of the tsetse fly and aphids. Moreover, by high-performance liquid chromatography, we measured the genomic G + C content of the S. oryzae principal endocytobiotes (SOPE) as 54%, while the known genomic G + C content of most intracellular bacteria is about 39.5%. Furthermore, based on the third codon position G + C content and the rrs gene G + C content, we demonstrated that most intracellular bacteria except SOPE are A + T biased irrespective of their phylogenetic position. Finally, using the hsp60 gene sequence, the codon usage of SOPE was compared with that of two phylogenetically closely related bacteria: E. coli, a free-living bacterium, and Buchnera aphidicola, the intracellular symbiotic bacteria of aphids. Taken together, these results show a peculiar and distinctly different DNA composition of SOPE with respect to the other obligate intracellular bacteria, and, combined with biological and biochemical data, they elucidate the evolution of symbiosis in S. oryzae.
Asunto(s)
Escarabajos/microbiología , ADN Bacteriano/química , Enterobacteriaceae/genética , Simbiosis , Animales , Composición de Base , Chaperonina 60/genética , ADN Ribosómico/química , Enterobacteriaceae/clasificación , Genes Bacterianos , Código Genético , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/química , Análisis de Secuencia de ADNRESUMEN
Specific proteins of symbiosis were analyzed by the comparison of two-dimensional electrophoresis protein patterns of symbiotic and aposymbiotic strains of the weevil Sitophilus oryzae. One protein was shown to be exclusively expressed in the aposymbiotic strain and three proteins, including a chaperonin, were characterized in the symbiotic strain pattern. The groE-like operon, encoding the two chaperonins groES and GroEL-like proteins of the endocytobiotes, was sequenced. It was found to be very similar to the groE operon of Escherichia coli (82% identity). In vitro and ex vivo experiments of protein labelling demonstrated that almost 40% of the endocytobiote protein synthesis ex vivo is focused on the GroEL-like protein. Finally, we showed by northern blotting that heat shock at 38 degrees C results in groEL mRNA accumulation inside the endocytobiotes. This work supports the hypothesis that chaperonins could have an essential physiological function in the maintenance of the symbiotic association.
Asunto(s)
Chaperonina 60/biosíntesis , Escarabajos/microbiología , Enterobacteriaceae/fisiología , Simbiosis , Secuencia de Aminoácidos , Animales , Chaperonina 10/química , Chaperonina 10/genética , Chaperonina 10/metabolismo , Chaperonina 60/química , Chaperonina 60/genética , Escarabajos/genética , Citosol/química , Citosol/microbiología , Electroforesis en Gel Bidimensional , Enterobacteriaceae/genética , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Simbiosis/genéticaRESUMEN
Chaperonins are ubiquitous proteins found in all prokaryotic and eukaryotic cells. They are overproduced in several parasitic bacteria and are implicated in at least 2 types of endocytobiosis: in amoebae and in aphids. This work puts in evidence that a protein named symbionin, which shows an immunological homology with the E. coli protein GroEL, is present in the symbiotic relationship of 3 species of Sitophilus (S. oryzae, S. granarius, and S. zeamais). This protein is neither found in the naturally asymbiotic specie S. linearis nor in the aposymbiotic strain of S. oryzae obtained in the laboratory. This symbionin is stored in a great quantity within endocytobiotes and its amino acid composition seems to corroborate its chaperonin resemblance rather than its possible function as one of the insect storage proteins already described in the literature.
Asunto(s)
Proteínas Bacterianas/análisis , Chaperoninas , Escarabajos/química , Endocitosis , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Chaperonina 60/química , Chaperonina 60/inmunología , Escherichia coli/química , Inmunohistoquímica , Análisis MultivarianteRESUMEN
In the Curculionid beetle Sitophilus oryzae, the fat body is composed of one type of adipocyte, interstitial cells and oenocytes. Synthesis and storage of tyrosine-rich-protein granules (TRPG) in adipocytes are observed during all the larval and prepupal stages (except the first larval instar which has not been studied). They appear first in the posterior part of the fat body, around the nucleus of adipocytes. They progressively invade the cytoplasm. In the young pupa, TRPG are present in every part of the body, including the head and the appendages in formation. TRPG grow in size by fusing together. Their mean diameter is 6 microm, but some of them reach up to 50 microm. They present a basic core and an acidic periphery. Their charge in tyrosine increases until the prepupa. They are APS and lipid negative and contain no RNA. During metamorphosis they take on a reticulated structure, evoking a golf ball, and disintegrate into small granules, the tyrosine content of which diminishes drastically, especially in contact with epidermal cells, whence tyrosine is probably transferred. TRPG in S. oryzae contain 16 different insoluble proteins. Five of them are tyrostaurins characterized by their very high content in tyrosine (up to 27%) and their strict insolubility in aqueous solution. Arylphorin-like proteins have not been detected in S. oryzae granules.
RESUMEN
The enzymatic activities of three lymphoblastoid cell lines were subjected to a comparative analysis of 22 enzymatic activities with a new microtechnique "Apizym". This method is easy, requires only a small number of cells and allows simultaneous testing of many enzymatic activities from the same sample. The three cell lines can be easily distinguished despite their close origin: NAB, derived from a malignant retroperitoneal mass in an American patient, is the cell line with the greatest total activity and HRlK (a clone of P3J, derived from an African lymphoma) with the lowest and RAJI (a non-virus producing line derived from an African lymphoma) being the intermediate. The more striking differences concern the activity of phosphatases (NAB greater than RAJI greater than or equal to HRlK), esterases-lipases (NAB greater than or equal to RAJI greater than HRlK), beta glucosaminidase, beta glucuronidase beta glucosidase (NAB greater than or equal to RAJI greater than HRlK). On the other hand, alpha mannosidase and alpha fucosidase are more active in RAJI (RAJI greater than NAB greater than or equal to HRlK).