Major outer membrane proteins with porin activity were isolated from cell envelopes of the halophilic strains Ectothiorhodospira shaposhnikovii N1 and Ectothiorhodospira vacuolata beta1. The porins were obtained as oligomers. They dissociated into monomers by heat or EDTA treatment. The molecular masses of the monomers were determined by mass spectrometry to be 39,285 and 37,160 Da for E. shaposhnikovii N1 and E. vacuolata beta1, respectively. Both were shown by analytical ultracentrifugation to be trimers of about 112, 000 Da. Circular dichroism spectra indicated predominantly beta-sheet structure. The 18 N-terminal amino acid sequences of the two porins were identical except for the amino acids in positions 12 and 14. No sequence similarity with the primary structure of known porins was found. In reconstitution experiments with lipid bilayers, the porins of E. shaposhnikovii N1 and E. vacuolata beta1 formed channels with a single-channel conductance of 1.5 and 0.7 nS, respectively, in 1 M KCl. The single-channel conductance saturated with increasing salt concentration, indicating a putative binding-site for anions in the channel since both porins exhibited anion-selectivity. For the porin of E. vacuolata beta1, but not for that of E. shaposhnikovii N1, an influence of detergent concentration on the single-channel conductance was observed.
ABSTRACT. Diatoms play a decisive role in global primary production, have a key role in the marine food web, and serve as food for the industrial culture of aquatic animals. In addition, species of diatoms have been chosen as tools for measuring sea water quality and for bioassay procedures. Among them, Phaeodactylum tricornutum (Bohlin) Lewin, 1958, represents a good biological model due to its ease of handling and also to its physiological, genetic and growth characteristics. We investigated the use of the Epifluorescence Microscopy (EPM) to visualize and enumerate P. tricornutum cells and bacteria in cultures containing both kinds of microorganisms. The cell suspension was filtered, stained with SYBR Green and analyzed by EPM. P. tricornutum can be enumerated using EPM with comparable accuracy as using a hemocytometer. The same kind of growth curve is obtained with either method. EPM is an advantageous alternative to the classic light microscope counting method; it allows the simultaneous enumeration of P. tricornutum and bacteria in a single sample, measuring red autofluorescence and green fluorescence respectively. Moreover, the methodology is suitable for dynamic studies performed to determine the growth dependence between P. tricornutum and bacteria. Another advantage of this technique is that different shapes, sizes and physiological stages of the microbiological communities can be observed.Key words: microscopic algae, bacteria, growth, enumeration, epifluorescence.Enumeración simultánea del crecimiento de Phaeodactylum tricornutum (MLB292) y bacterias en comunidades mixtas RESUMEN. Las diatomeas tienen un rol decisivo en la producción primaria global, un rol clave en la red trófica marina y sirven como alimento en el cultivo industrial de animales acuáticos. Algunas especies han sido elegidas para medir la calidad del agua y para bioensayos. Entre ellas, Phaeodactylum tricornutum (Bohlin) Lewin, 1958, representa un buen modelo biológico debido a su fácil manejo, como también a sus características fisiológicas, genéticas y de crecimiento. Se analiza el uso de la Microscopía de Epifluorescencia (MEP) para visualizar y enumerar células de P. tricornutum y bacterias en cultivos que contienen ambos tipos de microorganismos. Las células en suspensión fueron filtradas, marcadas con SYBR Green y analizadas con MEP. Las células de P. tricornutum se pueden contar con una precisión comparable a la del hemocitómetro y se obtiene la misma curva de crecimiento con ambos métodos. La MEP es una alternativa ventajosa al uso del clásico microscopio de luz, ya que permite el recuento simultáneo de P. tricornutum y bacterias en una misma muestra, midiéndose la autofluorescencia roja y la fluorescencia verde, respectivamente. Más aún, la metodología es apropiada para estudios de dinámica y para determinar la dependencia entre el crecimiento de P. tricornutum y las bacterias. Otra ventaja de esta técnica es que se puede observar diferencias de forma, tamaño y estado fisiológicos de las comunidades microbiológicas.Palabras ...
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