Cytofluorometric approaches to bacterial studies in aquatic environments

Memoria de tesis doctoral presentada por Tania Falcioni para obtener el título de Doctora por la Università degli Studi di Urbino "Carlo Bo" (UNIURB), realizada bajo la dirección del Dr. Josep Maria Gasol Piqué del Institut de Ciències del Mar (ICM-CSIC)Peer Reviewe

Evaluating the Flow-Cytometric Nucleic Acid Double-Staining Protocol in Realistic Situations of Planktonic Bacterial Death▿

Since heterotrophic prokaryotes play an important biogeochemical role in aquatic ecosystems and have a high capacity to survive in extreme environments, easy-to-perform protocols that probe their physiological states and the effects of environmental variables on those states are highly desired. Some methodologies combine a general nucleic acid stain with a membrane integrity probe. We calibrated one of these, the nucleic acid double-staining (NADS) protocol (G. Grégori, S. Citterio, A. Ghiani, M. Labra, S. Sgorbati, S. Brown, and M. Denis, Appl. Environ. Microbiol. 67:4662-4670, 2001), determining the optimal stain concentrations in seawater and the response to conditions that generate prokaryote death (such as heat) and to conditions that are known to produce death in plankton, such as nutrient limitation or flagellate grazing. The protocol was validated by comparison to two methods used to detect viability: active respiration by 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and incorporation of tritiated leucine. We show that concentrations in the range of 5 to 20 μg ml−1 of propidium iodide, simultaneous to a 10× concentration of Sybr green I, are best for detecting two separated populations of “live” (green cells) and “dead” (red cells) organisms. During exposure to heat and UVC, we observed that the number of live cells declined concurrently with that of actively respiring cells (CTC positive) and with total leucine incorporation. In seawater mesocosms, the NADS protocol allowed detection of bacterioplankton starvation-related death and flagellate predation. The protocol was also tested in deep profiles in the northwest Atlantic, demonstrating its potential for routine characterization of this fraction of the physiological diversity of marine heterotrophic prokaryotic plankton

Determination of the Viability of Aeromonas hydrophila in Different Types of Water by Flow Cytometry, and Comparison with Classical Methods

The presence of Aeromonas spp. in water can represent a risk for human health. Therefore, it is important to know the physiological status of these bacteria and their survival in the environment. We studied the behavior of a strain of Aeromonas hydrophila in river water, spring water, brackish water, mineral water, and chlorinated drinking water, which had different physical and chemical characteristics. The bacterial content was evaluated by spectrophotometric and plate count techniques. Flow cytometric determination of viability was carried out using a dual-staining technique that enabled us to distinguish viable bacteria from damaged and membrane-compromised bacteria. The traditional methods showed that the bacterial content was variable and dependent on the type of water. The results obtained from the plate count analysis correlated with the absorbance data. In contrast, the flow cytometric analysis results did not correlate with the results obtained by traditional methods; in fact, this technique showed that there were viable cells even when the optical density was low or no longer detectable and there was no plate count value. According to our results, flow cytometry is a suitable method for assessing the viability of bacteria in water samples. Furthermore, it permits fast detection of bacteria that are in a viable but nonculturable state, which are not detectable by conventional methods

Virion Stability Is Important for the Circulative Transmission of Tomato Yellow Leaf Curl Sardinia Virus by Bemisia tabaci, but Virion Access to Salivary Glands Does Not Guarantee Transmissibility▿ †

The capsid protein (CP) of the monopartite begomovirus Tomato yellow leaf curl Sardinia virus (TYLCSV), family Geminiviridae, is indispensable for plant infection and vector transmission. A region between amino acids 129 and 152 is critical for virion assembly and insect transmissibility. Two previously described mutants, one with a double Q129P Q134H mutation (PNHD) and another with a further D152E change (PNHE), were found nontransmissible (NT). Another NT mutant with a single N130D change (QDQD) was retrieved from a new mutational analysis. In this study, these three NT mutants and the wild-type (wt) virus were compared in their relationships with the whitefly vector Bemisia tabaci and the nonvector Trialeurodes vaporariorum. Retention kinetics of NT mutants were analyzed by quantitative dot blot hybridization in whiteflies fed on infected plants. The QDQD mutant, whose virions appeared nongeminate following purification, was hardly detectable in either whitefly species at any sampling time. The PNHD mutant was acquired and circulated in both whitefly species for up to 10 days, like the wt virus, while PNHE circulated in B. tabaci only. Using immunogold labeling, both PNHD and PNHE CPs were detected in B. tabaci salivary glands (SGs) like the wt virus, while no labeling was found in any whitefly tissue with the QDQD mutant. Significant inhibition of transmission of the wt virus was observed after prior feeding of the insects on plants infected with the PNHE mutant, but not on plants infected with the other mutants. Virion stability and ability to cross the SG barrier are necessary for TYLCSV transmission, but interactions with molecular components inside the SGs are also critical for transmissibility

Salicylic acid mitigates physiological and proteomic changes induced by the SPCP1 strain of Potato virus X in tomato plants

Induction of resistance by salicylic acid (SA) exogenous treatment is a complementary approach to control plant diseases. SA effect on Potato virus X (SPCP1 strain) - infected tomato plants was examined by analyzing their physiological parameters and proteomic profiling at initial infection. PVX-SPCP1 altered photosynthesis and carbohydrate synthesis proteins and elicited stress proteins. SA partially offset reduction in photosynthetic rate during infection by increasing mesophyll conductance. SA counteracted these changes through stabilization of photosystem II, increased proteins related with thermotolerance and stress, and decreased proteins related with stomatal opening. The strongest effects of SA occurred at the beginning of the pathogenesis cycle. (C) 2015 Elsevier Ltd. All rights reserved

Salicylic acid mitigates physiological and proteomic changes induced by the SPCP1 strain of Potato virus X in tomato plants

Induction of resistance by salicylic acid (SA) exogenous treatment is a complementary approach to control plant diseases. SA effect on Potato virus X (SPCP1 strain) - infected tomato plants was examined by analyzing their physiological parameters and proteomic profiling at initial infection. PVX-SPCP1 altered photosynthesis and carbohydrate synthesis proteins and elicited stress proteins. SA partially offset reduction in photosynthetic rate during infection by increasing mesophyll conductance. SA counteracted these changes through stabilization of photosystem II, increased proteins related with thermotolerance and stress, and decreased proteins related with stomatal opening. The strongest effects of SA occurred at the beginning of the pathogenesis cycle