RESUMEN
Coral bleaching is a disease that threatens coral reefs throughout the world. The disease is correlated with higher-than-normal seawater temperatures. Data have been reported showing that bleaching of the coral Oculina patagonica during the summer in the Mediterranean Sea is the result of an infection with Vibrio shiloi. The summer temperatures induce the expression of virulence factors in the pathogen. We report here that V. shiloi produces an extracellular superoxide dismutase (SOD) at 30 degrees C, but not at 16 degrees C. An SOD(-) mutant was avirulent. The mutant adhered to corals, penetrated into coral cells, multiplied intracellularly for a short time, and then died. These data support the hypothesis that SOD protects the intracellular V. shiloi from oxidative stress caused by the high concentration of oxygen produced by intracellular zooxanthellae photosynthesis.
Asunto(s)
Antozoos/microbiología , Superóxido Dismutasa/biosíntesis , Vibrio/enzimología , Factores de Virulencia/biosíntesis , Eucariontes/metabolismo , Biblioteca de Genes , Mar Mediterráneo , Mutagénesis Insercional , Estaciones del Año , Agua de Mar , Superóxido Dismutasa/genética , Superóxido Dismutasa/aislamiento & purificación , Simbiosis , Temperatura , Vibrio/patogenicidadRESUMEN
The bioemulsifier of Acinetobacter radioresistens KA53, referred to as alasan, is a high-molecular-weight complex of polysaccharide and protein. Recently, one of the alasan proteins, with an apparent molecular mass of 45 kDa, was purified and shown to constitute most of the emulsifying activity. The N-terminal sequence of the 45-kDa protein showed high homology to an OmpA-like protein from Acinetobacter spp. In the research described here the gene coding for the 45-kDa protein was cloned, sequenced, and expressed in Escherichia coli. Recombinant protein AlnA (35.77 kDa without the leader sequence) had an amino acid sequence homologous to that of E. coli OmpA and contained 70% of the specific (hydrocarbon-in-water) emulsifying activity of the native 45-kDa protein and 2.4 times that of the alasan complex. In addition to their emulsifying activity, both the native 45-kDa protein and the recombinant AlnA were highly effective in solubilizing phenanthrene, ca. 80 microg per mg of protein, corresponding to 15 to 19 molecules of phenanthrene per molecule of protein. E. coli OmpA had no significant emulsifying or phenanthrene-solubilizing activity. The production of a recombinant surface-active protein (emulsification and solubilization of hydrocarbons in water) from a defined gene makes possible for the first time structure-function studies of a bioemulsan.
Asunto(s)
Acinetobacter/genética , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Excipientes/aislamiento & purificación , Polisacáridos Bacterianos/genética , Acinetobacter/química , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/biosíntesis , Proteínas Bacterianas/química , Datos de Secuencia Molecular , Fenantrenos , Polisacáridos Bacterianos/química , Proteínas Recombinantes/biosíntesis , Homología de Secuencia de Aminoácido , SolubilidadRESUMEN
The antibiotic TA of Myxococcus xanthus is a complex macrocyclic polyketide, produced through successive condensations of acetate by a type I PKS (polyketide synthase) mechanism. The genes encoding TA biosynthesis are clustered on a 36 kb DNA fragment, which has been cloned and analysed. The chemical structure of TA and the mechanism by which it is synthesized indicate the need for several post-modification steps, which are introduced into the carbon chain of the polyketide to form the final bioactive molecule. These include the addition of several carbon atoms originating from acetate carbonyl, three C-methylations, O-methylation and a specific hydroxylation. This paper reports the analysis of five genes which are involved in the post-modification of TA. Their functional analysis, by specific gene disruption, suggests that they may be essential for the production of the active antibiotic. The characteristics and organization of the genes suggest that they may be involved in the addition of the carbon atoms which arise from acetate.
Asunto(s)
Antibacterianos/metabolismo , Genes Bacterianos/fisiología , Complejos Multienzimáticos/genética , Myxococcus xanthus/genética , Secuencia de Aminoácidos , Antibacterianos/química , Secuencia de Bases , Expresión Génica/genética , Macrólidos , Datos de Secuencia Molecular , Familia de Multigenes/genética , Myxococcus xanthus/metabolismo , Sistemas de Lectura Abierta/genética , Sistemas de Lectura Abierta/fisiología , Procesamiento Proteico-Postraduccional/genética , Procesamiento Proteico-Postraduccional/fisiología , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
DNA gyrase, an essential type II topoisomerase, mediates negative supercoiling of the bacterial chromosome, thereby affecting the processes of DNA replication, transcription, recombination and repair. The gyrB gene from the Gram-negative soil bacterium Myxococcus xanthus was sequenced. The sequence predicts a protein of 815 amino acid residues displaying significant homology to all known GyrB proteins. A 6-His-GyrB fusion protein was overexpressed in Escherichia coli and purified to near homogeneity using affinity chromatography on Ni-nitrilotriacetic acid-agarose and novobiocin-Sepharose columns. The fusion protein bound novobiocin and cross-reacted with anti-E. coli GyrB antibodies, indicating structural and functional similarities to the E. coli DNA GyrB. The gene was mapped to the region of the origin of replication (oriC) of M. xanthus.
