Coronin Is a Component of the Endocytic Collar of Hyphae of Neurospora crassa and Is Necessary for Normal Growth and Morphogenesis

Coronin plays a major role in the organization and dynamics of actin in yeast. To investigate the role of coronin in a filamentous fungus (Neurospora crassa), we examined its subcellular localization using fluorescent proteins and the phenotypic consequences of coronin gene (crn-1) deletion in hyphal morphogenesis, Spitzenkörper behavior and endocytosis. Coronin-GFP was localized in patches, forming a subapical collar near the hyphal apex; significantly, it was absent from the apex. The subapical patches of coronin colocalized with fimbrin, Arp2/3 complex, and actin, altogether comprising the endocytic collar. Deletion of crn-1 resulted in reduced hyphal growth rates, distorted hyphal morphology, uneven wall thickness, and delayed establishment of polarity during germination; it also affected growth directionality and increased branching. The Spitzenkörper of Δcrn-1 mutant was unstable; it appeared and disappeared intermittently giving rise to periods of hyphoid-like and isotropic growth respectively. Uptake of FM4-64 in Δcrn-1 mutant indicated a partial disruption in endocytosis. These observations underscore coronin as an important component of F-actin remodeling in N. crassa. Although coronin is not essential in this fungus, its deletion influenced negatively the operation of the actin cytoskeleton involved in the orderly deployment of the apical growth apparatus, thus preventing normal hyphal growth and morphogenesis

Short-term variability in the activity and composition of the diazotroph community in a coastal upwelling system

Today we know that diazotrophs are common and active in nitrogen (N) replete regions, however the factors controlling their distribution remain elusive. Previous studies in upwelling regions revealed that the composition of diazotrophs responded to changes in hydrodynamic forcing over seasonal scales. Here we used high-frequency observations collected during a 3-week cruise in the upwelling region off NW Iberia to describe changes in the activity and composition of diazotrophs over shorter temporal scales. The cruise started after a strong upwelling event followed by a few days of relaxation-downwelling, and soon after another upwelling pulse. Higher N2 fixation rates (2.2 ± 0.7 µmol m-3 d-1) were measured during relaxation-downwelling, when surface nitrate concentration was low. During the fertilization associated with the upwelling, N2 fixation dramatically decreased to 0.10 ± 0.09 µmol m-3 d-1. The comparison with nitrate consumption and diffusion confirmed the minor role of N2 fixation (<1%) as a source of new N for primary production. The unicellular cyanobacterium UCYN-A2 was the dominant diazotroph during the cruise. UCYN-A2 abundance was four times higher during relaxation-downwelling (4x104 copies L-1) compared to upwelling conditions (0.2x104 copies L-1), when the unusual Epsilonproteobacteria increased their relative abundance. These results indicate that diazotrophs can respond rapidly to changes in the environment, and point out to the availability of N as a key factor controlling the activity, composition and distribution of diazotrophs in eutrophic regions

Hydrography of the Pontevedra Ria: Intra-annual spatial and temporal variability in a Galician coastal system (NW Spain)

In order to ameliorate the dearth of existing scientific knowledge concerning the hydrography of the Pontevedra Ria, a systematic investigation was carried out between October 1997-98. Salinity variations were closely related to river discharge whereas bottom waters presented oceanic characteristics over the whole year. Current was controlled by tide, river discharge, and wind in the internal ria where the highest velocities were directed along the ria channel with a low transverse component. Favorable atmospheric conditions in spring induced coastal upwelling up the continental shelf. In May the upwelling was sufficiently strong to be detected in the inner ria and intensified in July and August, cooling the ria water to 12 degrees -14 degreesC. Upwelling ceased in September, and from November to March seawater transported by the poleward current (35.9; 15 degreesC) was detected on the shelf. From January until March, unanticipated favorable upwelling conditions provoked an influx of poleward inside the ria. Ria intrusion of poleward water and association with occasional winter upwelling conditions has not been observed previously. Isopycnic three-dimensional (3-D) surface and 2-D isopycnal maps show that with high river runoff or intense upwelling, lower-salinity water leaves the ria near the northern margin in the surface layer. Under negative upwelling conditions, the water is partially dammed inside the ria and exits the ria when the wind speed falls. During upwelling events, ENACW penetrated the ria, especially near the southern shore. Arrival of ENACW at the northern entrance impedes the outward water flow through this mouth

Growth of Vibrio cholerae O1 in Red Tide Waters off California

Vibrio cholerae serotype O1 is autochthonous to estuarine and coastal waters. However, its population dynamics in such environments are not well understood. We tested the proliferation of V. cholerae N16961 during a Lingulodinium polyedrum bloom, as well as other seawater conditions. Microcosms containing 100-kDa-filtered seawater were inoculated with V. cholerae or the 0.6-μm-pore-size filterable fraction of seawater assemblages. These cultures were diluted 10-fold with fresh 100-kDa-filtered seawater every 48 h for four cycles. Growth rates ranged from 0.3 to 14.3 day(−1) (4.2 day(−1) ± 3.9) for V. cholerae and 0.1 to 9.7 day(−1) (2.2 ± 2.8 day(−1)) for bacterial assemblage. Our results suggest that dissolved organic matter during intense phytoplankton blooms has the potential to support explosive growth of V. cholerae in seawater. Under the conditions tested, free-living V. cholerae was able to reach concentrations per milliliter that were up to 3 orders of magnitude higher than the known minimum infectious dose (10(4) cell ml(−1)) and remained viable under many conditions. If applicable to the complex conditions in marine ecosystems, our results suggest an important role of the growth of free-living V. cholerae in disease propagation and prevention during phytoplankton blooms

Localization and role of MYO-1, an endocytic protein in hyphae of Neurospora crassa.

The subapical endocytic collar is a prominent feature of hyphae of Neurospora crassa. It comprises a dynamic collection of actin patches associated with a number of proteins required for endocytosis, namely, ARP-2/3 complex, fimbrin, coronin, etc. We presently show that MYO-1 is another key component of this endocytic collar. A myo-1 sequence was identified in the genome of N. crassa and used it to generate a strain with a myo-1-sgfp allele under the ccg1 promoter. Examination of living hyphae by confocal microscopy, revealed MYO-1-GFP located mainly as a dynamic collection of small patches arranged in collar-like fashion in the hyphal subapex. Dual tagging showed MYO-1-GFP partially colocalized with two other endocytic proteins, fimbrin and coronin. MYO-1 was also present during septum formation. By recovering a viable strain, albeit severely inhibited, after deletion of myo-1, it was possible to investigate the phenotypic consequences of the elimination of MYO-1. Deletion of myo-1 caused a severe reduction in growth rate (95%), near absence of aerial mycelium and no conidiation. A reduced uptake of the lipophilic dye FM4-64 indicated a deficiency in endocytosis in the Δmyo-1 mutant. Hyphae were produced by the Δmyo-1 mutant but their morphogenesis was severely affected; hyphal morphology was distorted displaying irregular periods of isotropic and polarized growth. The morphological alterations were accompanied, and presumably caused, by a disruption in the organization and dynamics of a myosin-deprived actin cytoskeleton that, ultimately, compromised the stability and function of the Spitzenkörper as a vesicle supply center

Localization and role of MYO-1, an endocytic protein in hyphae of Neurospora crassa.

The subapical endocytic collar is a prominent feature of hyphae of Neurospora crassa. It comprises a dynamic collection of actin patches associated with a number of proteins required for endocytosis, namely, ARP-2/3 complex, fimbrin, coronin, etc. We presently show that MYO-1 is another key component of this endocytic collar. A myo-1 sequence was identified in the genome of N. crassa and used it to generate a strain with a myo-1-sgfp allele under the ccg1 promoter. Examination of living hyphae by confocal microscopy, revealed MYO-1-GFP located mainly as a dynamic collection of small patches arranged in collar-like fashion in the hyphal subapex. Dual tagging showed MYO-1-GFP partially colocalized with two other endocytic proteins, fimbrin and coronin. MYO-1 was also present during septum formation. By recovering a viable strain, albeit severely inhibited, after deletion of myo-1, it was possible to investigate the phenotypic consequences of the elimination of MYO-1. Deletion of myo-1 caused a severe reduction in growth rate (95%), near absence of aerial mycelium and no conidiation. A reduced uptake of the lipophilic dye FM4-64 indicated a deficiency in endocytosis in the Δmyo-1 mutant. Hyphae were produced by the Δmyo-1 mutant but their morphogenesis was severely affected; hyphal morphology was distorted displaying irregular periods of isotropic and polarized growth. The morphological alterations were accompanied, and presumably caused, by a disruption in the organization and dynamics of a myosin-deprived actin cytoskeleton that, ultimately, compromised the stability and function of the Spitzenkörper as a vesicle supply center

Correlación digital del color para el reconocimiento de Vibrio cholerae 01 en muestras ambientales y de laboratorio

En general, el conteo directo refleja más la abundancia microbiológica que el conteo de placas. Se han desarrollado técnicas microbiológicas para reconocer y contar microorganismos en sistemas naturales pero han tenido problemas con la confiabilidad de sus resultados. Frecuentemente, métodos distintos dan diferentes resultados para la enumeración de un organismo específico. Para la examinación directa al microscopio de bacterias, los sistemas digitales automatizados representan un posible avance en la identificación y conteo, eliminando una predisposición del observador y reduciendo costos y tiempo de análisis. En este trabajo se desarrolló un programa computacional para evaluar la utilidad de sistemas ópticos coherentes para el reconocimiento de Vibrio cholerae 01, mediante correlación de color en cultivos de laboratorio y de muestras ambientales teñidas con anticuerpos monoclonales fluorescentes. De muestras de laboratorio probamos 94 muestras positivas y 115 negativas y 33 muestras positivas y 34 negativas de muestras ambientales y 613 muestras positivas y 546 negativas de muestras de mesocosmos. En muestras de laboratorio se llevó a cabo una correcta identificación, un conteo de células y una discriminación a un 100%. La sensibilidad del sistema digital en muestras ambientales como en muestras análogas al ambiente varió de 91% a 94% y tuvo un 99,5% de discriminación entre otras bacterias o partículas. Basados en los valores absolutos de correlación en los componentes rojo, verde y azul de las imágenes policromáticas de V. cholerae 01 (canales RGB), el algorítmo para contar e identificar correlacionó bien con los picos en la salida del canal verde y no hubo picos de salida en los canales rojo y azul. El criterio de discriminación correlacionó bien con los picos presentes en los canales rojo y azul. Concluímos que el sistema digital de correlación de color para identificar y contar V. cholerae 01 de muestras ambientales y de laboratorio es una herramienta útil con alta confiabilidad

Correlación digital del color para el reconocimiento de Vibrio cholerae 01 en muestras ambientales y de laboratorio

En general, el conteo directo refleja más la abundancia microbiológica que el conteo de placas. Se han desarrollado técnicas microbiológicas para reconocer y contar microorganismos en sistemas naturales pero han tenido problemas con la confiabilidad de sus resultados. Frecuentemente, métodos distintos dan diferentes resultados para la enumeración de un organismo específico. Para la examinación directa al microscopio de bacterias, los sistemas digitales automatizados representan un posible avance en la identificación y conteo, eliminando una predisposición del observador y reduciendo costos y tiempo de análisis. En este trabajo se desarrolló un programa computacional para evaluar la utilidad de sistemas ópticos coherentes para el reconocimiento de Vibrio cholerae 01, mediante correlación de color en cultivos de laboratorio y de muestras ambientales teñidas con anticuerpos monoclonales fluorescentes. De muestras de laboratorio probamos 94 muestras positivas y 115 negativas y 33 muestras positivas y 34 negativas de muestras ambientales y 613 muestras positivas y 546 negativas de muestras de mesocosmos. En muestras de laboratorio se llevó a cabo una correcta identificación, un conteo de células y una discriminación a un 100%. La sensibilidad del sistema digital en muestras ambientales como en muestras análogas al ambiente varió de 91% a 94% y tuvo un 99,5% de discriminación entre otras bacterias o partículas. Basados en los valores absolutos de correlación en los componentes rojo, verde y azul de las imágenes policromáticas de V. cholerae 01 (canales RGB), el algorítmo para contar e identificar correlacionó bien con los picos en la salida del canal verde y no hubo picos de salida en los canales rojo y azul. El criterio de discriminación correlacionó bien con los picos presentes en los canales rojo y azul. Concluímos que el sistema digital de correlación de color para identificar y contar V. cholerae 01 de muestras ambientales y de laboratorio es una herramienta útil con alta confiabilidad