Abstract
Isolates of the marine picocyanobacteria, Prochlorococcus and Synechococcus, are often accompanied by diverse heterotrophic “contaminating” bacteria, which can act as confounding variables in otherwise controlled experiments. Traditional microbiological methods for eliminating contaminants, such as direct streak-plating, are often unsuccessful with this particular group of microorganisms. While they will grow in pour plates, colonies often remain contaminated with heterotrophic bacteria that can migrate through the soft agar. Additionally, axenic clones of picocyanobacteria can be recovered via dilution-to-extinction in liquid medium, but the efficiency of recovery is low, often requiring large numbers of 96-well plates. Here, we detail a simple and effective protocol for rendering cultures of Synechococcus and Prochlorococcus strains free of bacterial contaminants while at the same time yielding clonal isolates. We build on the fact that co-culture with specific heterotrophs—“helper heterotrophs”—is often necessary to grow colonies of picocyanobacteria from single cells in agar. Suspecting that direct physical contact between the helper and the picocyanobacterial cells was not necessary for the “helper effect,” we developed a protocol in which the helper cells are embedded in soft agar pour plates, a filter overlaid on the surface, and a picocyanobacterial culture is diluted and then spotted on top of the filter. With this approach, motile contaminants cannot swim to the colonies, and it is possible to obtain the expected number of colonies from a given input (i.e., a Poisson distribution of colonies with an expected value equal to the input number of cells), thus ensuring clonal colonies. Using this protocol, we rendered three strains of Synechococcus, two strains of Prochlorococcus, and 19 new strains of Synechococcus from coastal seawater clonal and free of heterotrophic bacteria. The simplicity of this approach should expand the repertoire of axenic picocyanobacterial strains available for controlled physiological experiments. It will also enable the study of microdiversity in populations of picocyanobacteria by facilitating large-scale isolation of picocyanobacterial clones from a single source, including direct isolation from natural seawater.
Highlights
Marine picocyanobacteria, which are at the base of marine microbial food webs, are frequently brought into culture using enrichments on nutrient-amended raw or filtered seawater, or by using dilution-to-extinction approaches (Waterbury and Stanier, 1981; Waterbury, 1986; Moore et al, 2002, 2007; Ahlgren and Rocap, 2006)
The filter plating protocol described here will produce individually arrayed clones of picocyanobacteria that are free of heterotrophic bacterial contaminants after a series of plating techniques involving helper bacteria, and downstream verifying techniques (Figure 1)
It is possible for clones to be contaminated with heterotrophic bacteria, but the remaining heterotroph(s) can typically be removed by a secondary round of purification via filter plating
Summary
Marine picocyanobacteria, which are at the base of marine microbial food webs, are frequently brought into culture using enrichments on nutrient-amended raw or filtered seawater, or by using dilution-to-extinction approaches (Waterbury and Stanier, 1981; Waterbury, 1986; Moore et al, 2002, 2007; Ahlgren and Rocap, 2006) These cultures are rarely (if ever) free of heterotrophic bacterial contaminants, due to the basal carbon in seawater and production of fixed organic carbon by the phototrophs that support these contaminants. Heterotrophs can affect coarse-grained parameters like growth rate for Prochlorococcus (Morris et al, 2011), finegrained parameters like gene expression (Biller et al, 2016), susceptibility to stressors (e.g., light, temperature, and pH; Ma et al, 2017), or interactions with other biotic factors (viruses or bacterivores), so obtaining contaminant-free cultures is key to advancing these picocyanobacteria as model systems (AguiloFerretjans et al, 2021) They are often not purified out of culture because removing them is such a significant challenge. Clonal cultures remain contaminated with heterotrophic bacteria—likely because heterotrophs can swim through soft agar toward the phototrophs
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