Search

SinigallianoC.D., WinshellJ., GuerreroM.A., ScorzettiG., FellJ.W., EatonR.W., BrandL. andReinK.S. 2009. Viable cell sorting of dinoflagellates by multiparametric flow cytometry. Phycologia48: 249–257. DOI: 10.2216/08-51.1.Electronic cell sorting for isolation and culture of dinoflagellates and other marine eukaryotic phytoplankton was compared to the traditional method of manually picking cells using a micropipette. Trauma to electronically sorted cells was not a limiting factor, as fragile dinoflagellates, such as Karenia brevis(Dinophyceae), survived electronic cell sorting to yield viable cells. The rate of successful isolation of large-scale (> 4 litres) cultures was higher for manual picking than for electronic cell sorting (2% vs 0.5%, respectively). However, manual picking of cells is more labor intensive and time consuming. Most manually isolated cells required repicking, as the cultures were determined not to be unialgal after a single round of isolation; whereas, no cultures obtained in this study from electronic single-cell sorting required resorting. A broad flow cytometric gating logic was employed to enhance species diversity. The percentages of unique genotypes produced by manual picking or electronic cell sorting were similar (57% vs 54%, respectively), and each approach produced a variety of dinoflagellate or raphidophyte genera. Alternatively, a highly restrictive gating logic was successfully used to target K. brevisfrom a natural bloom sample. Direct electronic single-cell sorting was more successful than utilizing a pre-enrichment sort followed by electronic single-cell sorting. The appropriate recovery medium may enhance the rate of successful isolations. Seventy percent of isolated cells were recovered in a new medium (RE) reported here, which was optimized for axenic dinoflagellate cultures. The greatest limiting factor to the throughput of electronic cell sorting is the need for manual postsort culture maintenance and assessment of the large number of isolated cells. However, when combined with newly developed automated methods for growth screening, electronic single-cell sorting has the potential to accelerate the discovery of new algal strains.

Soil isolates of Pseudomonas stutzeri have been shown previously to acquire genes by natural transformation. In this study a marine isolate, Pseudomonas stutzeri strain ZoBell, formerly Pseudomonas perfectomarina, was also shown to transform naturally. Transformation was detected by the Juni plate method and frequencies of transformation were determined by filter transformation procedures. Maximum frequencies of transformation were detected for three independent antibiotic resistance loci. Transformation frequencies were on the order of 4×10-5 transformants per recipient, a frequency over 100 times that of spontancous antibiotic resistance. Transfer of antibiotic resistance was inhibited by DNase I digestion. Marine isolates achieved maximum competence 14 h after transfer of exponential cultures to filters on solid media, although lower levels of competence were detected immediately following filter immobilization. Like soil isolates, P. stutzeri strain ZoBell is capable of cell contact transformation, but unlike soil isolates where transformation frequencies are greater for cell contact transformation as compared to transformation with purified DNA, the maximum frequency of transformation achieved by cell contact in the marine strain was approximately 10-fold less than transformation frequencies with purified DNA. These studies establish the first marine model for the study of natural transformation.

Both the soil isolate,Pseudomonas stutzeri JM300, and the marine isolate,Pseudomonas stutzeri strain ZoBell, have been shown previously to be naturally transformable. This study reports the detection of genetic exchange by natural transformation between these two isolates. Transformation frequency was determined by filter transformation procedures. Three independent antibiotic resistance loci were used as chromosomal markers to monitor this exchange event: resistance to rifampicin, streptomycin, and nalidixic acid. The maximum frequencies of transformation were on the order of 3.1 to 3.8×10-6 transformants per recipient; frequencies over an order of magnitude greater than those for spontaneous antibiotic resistance, although they are lower than those observed for soil: soil or marine: marine strain crosses. This exchange was inhibited by DNase I. Transformation was observed between soil and marine strains, both by filter transformation using purified DNA solutions and when transforming DNA was added in the form of viable donor cells. The results from this study support the close genetic relationship betweenP. stutzeri JM300 andP. stutzeri strain ZoBell. These results also further validate the utility ofP. stutzeri as a benchmark organism for modeling gene transfer by natural transformation in both soil and marine habitats.

The capacity of marine sediments to facilitate natural transformation of Pseudomonas stutzeri strain ZoBell was investigated. The role of DNA/ sediment binding on transformation frequency was also explored. While transformation frequencies increased as a function of DNA concentrations from 0 to 1.0 µg of DNA from rifampin-resistant strains for filter transformations, transformation in autoclaved sediment columns displayed a reduction in transformation frequency in response to low DNA concentrations (0–1.0 µg/cm3 sediment). Maximum transformation frequencies were obtained on filters at 1.0 µg exogenous DNA, however, maximum frequencies were not reached in sediments until a DNA concentration of 3.0 µg/cm3 sediment was added. When autoclaved sediments were pre-loaded with excess calf thymus DNA, this reduction in response of transformation frequency (cf. filter transformation) was eliminated, i.e., transformation frequencies reached saturation in the preloaded sediments at 1.0 µg DNA/cm3. Autoclaved sediments were shown to bind DNA at a concentration of about 3.6 µg/cm3 sediment, and maximum transformation frequencies were only obtained when these sediments were saturated with DNA. These data indicate that autoclaved marine sediments have the capacity to bind DNA in a form that prevents its availability for natural transformation and only after sediments are saturated with DNA does exogenous DNA become biologically active for natural transformation.