Persistence of Only a Minute Viable Population in Chlorotic Microcystis aeruginosa PCC 7806 Cultures Obtained by Nutrient Limitation

RESEARCH ARTICLE Persistence of Only a Minute Viable Population in Chlorotic Microcystis aeruginosa PCC 7806 Cultures Obtained by Nutrient Limitation Diogo de Abreu Meireles1¤, Jan Schripsema2, Andrea Cristina Vetö Arnholdt3, Denise Dagnino1,2* 1 Laboratório de Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, Rio de Janeiro, Brazil, 2 Grupo Metabolômica, Laboratório de Ciências Químicas, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, Rio de Janeiro, Brazil, 3 Laboratório de Biologia do Reconhecer, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, Rio de Janeiro, Brazil ¤ Current Address: Departamento Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil * OPEN ACCESS Citation: de Abreu Meireles D, Schripsema J, Vetö Arnholdt AC, Dagnino D (2015) Persistence of Only a Minute Viable Population in Chlorotic Microcystis aeruginosa PCC 7806 Cultures Obtained by Nutrient Limitation. PLoS ONE 10(7): e0133075. doi:10.1371/ journal.pone.0133075 Editor: Ivan Berg, University of Freiburg, GERMANY Received: April 8, 2015 Accepted: June 22, 2015 Published: July 16, 2015 Copyright: © 2015 Meireles et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The authors thank the Conselho Nacional de Pesquisa (CNPq) and the Fundação de Amparo à Pesquisa do Rio de Janeiro (FAPERJ) for financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abstract Cultures from the cyanobacterial strain Microcystis aeruginosa PCC 7806 submitted to nutrient limitation become chlorotic. When returned to nutrient rich conditions these cultures regain their green colour. The aim of this study was to verify whether the cells in these cultures could be considered resting stages allowing the survival of periods of nutrient starvation as has been reported for Synechococcus PCC 7942. The experiments with Microcystis were carried out in parallel with Synechococcus cultures to rule out the possibility that any results obtained with Microcystis were due to our particular experimental conditions. The results of the experiments with Synechococcus PCC 7942 cultures were comparable to the reported in the literature. For Microcystis PCC 7806 a different response was observed. Analysis of chlorotic Microcystis cultures by flow cytometry showed that the phenotype of the cells in the population was not homogenous: the amount of nucleic acids was about the same in all cells but only around one percent of the population emitted red autofluorescence indicating the presence of chlorophyll. Monitoring of the reversion of chlorosis by flow cytometry showed that the re-greening was most likely the result of the division of the small population of red autofluorescent cells originally present in the chlorotic cultures. This assumption was confirmed by analysing the integrity of the DNA and the membrane permeability of the cells of chlorotic cultures. Most of the DNA of these cultures was degraded and only the autofluorescent population of the chlorotic cultures showed membrane integrity. Thus, contrary to what has been reported for other cyanobacterial genera, most of the cells in chlorotic Microcystis cultures are not resting stages but dead. It is interesting to note that the red autofluorescent cells of green and chlorotic cultures obtained in double strength ASM-1 medium differ with respect to metabolism: levels of emission of red autofluorescence are higher in the cells of green cultures and the ability to convert fluorescein diacetate of PLOS ONE | DOI:10.1371/journal.pone.0133075 July 16, 2015 1 / 17 Survival of Minute Population of Microcystis under Nutrient Limitation these cells are heterogeneous when compared to the autofluorescent cells of chlorotic cultures. Thus, the small population of the red autofluorescent cells of chlorotic cultures are in a differentiated metabolic state that allow them to persist in conditions in which most of the population loses viability; persistent cells can be detected in chlorotic cultures maintained for more than a year. Introduction The cyanobacteria form a monophyletic group comprising the oxygenic photosynthetic bacteria [1]. They are inhabitants of many types of environments and comprise organisms capable of living under extreme conditions of temperature, radiation intensity and water availability [2], [3] and [4]. Cyanobacteria are among the few organisms able to inhabit several of the earth’s most extreme environments like the core of the Atacama Desert, arctic deserts and hot springs. Usual inhabitants of extreme environments, under certain unfavourable circumstances for growth, specialised resting cells (akinetes) can develop from vegetative cells. Like the spores of other bacteria, when compared to vegetative cells, akinetes are more resistant to environmental stresses. Akinetes have been shown to be resistant to drying, freezing and long-term storage in anoxic sediments [1]. Adaptations of vegetative cells that allow cyanobacteria to survive inadequate growth conditions have also been reported and include desiccation tolerance and chlorosis (sometimes called bleaching). Several genera of cyanobacteria are resistant to desiccation [2]. Under these circumstances cell structure is maintained, compatible solutes are accumulated [5] while metabolism decreases to undetectable levels. Rehydration leads to a rapid return of metabolic activity [6]. Chlorosis is the other often reported adaptation typical for vegetative cells of cyanobacteria to inadequate nutrient availability, while exposed to light. It occurs by the dismantling of the photosynthetic apparatus [7]. Cultures of several genera respond to this inadequate growth condition by acquiring a chlorotic phenotype [8], [9] and [10]. These studies show that reversion of chlorosis can be achieved once the cultures are returned to adequate culture conditions. The reversion of chlorosis has been particularly investigated in Synechococcus PCC 7942 cultures. In these chlorotic cultures photosynthesis was around 0.1% [11] of the photosynthesis reported for green cultures. When returned to adequate growth conditions nearly all cells of the chlorotic cultures regained their red autofluorescence and divided after 4–5 days of incubation [12]. Microcystis is often reported as responsible for toxic blooms [13]. It has a complex life cycle that allows it to withstand environmental variations [14]. During spring and su (...truncated)