Facilitation of Robust Growth of Prochlorococcus Colonies and Dilute Liquid Cultures by “Helper” Heterotrophic Bacteria

Abstract

Axenic (pure) cultures of marine unicellular cyanobacteria of the Prochlorococcus genus grow efficiently only if the inoculation concentration is large; colonies form on semisolid medium at low efficiencies. In this work, we describe a novel method for growing Prochlorococcus colonies on semisolid agar that improves the level of recovery to approximately 100%. Prochlorococcus grows robustly at low cell concentrations, in liquid or on solid medium, when cocultured with marine heterotrophic bacteria. Once the Prochlorococcus cell concentration surpasses a critical threshold, the "helper" heterotrophs can be eliminated with antibiotics to produce axenic cultures. Our preliminary evidence suggests that one mechanism by which the heterotrophs help Prochlorococcus is the reduction of oxidative stress.Members of the genus Prochlorococcus are the most abundant marine photosynthetic organisms and, as such, are major contributors to photosynthesis in the ocean (20). Over 30 strains of Prochlorococcus have been brought into culture, isolated from many locations within the band from 40°N to 40°S, including the North Atlantic, the North and South Pacific Oceans, the Mediterranean Sea, and the Arabian Sea (20). Despite this success, very few pure cultures of Prochlorococcus (e.g., those of strains PCC 9511 and MIT 9313 [18,22]) have been obtained. The vast majority of cultures contain heterotrophic microbes as contaminants; these heterotrophs were cocultured from the marine environment during the isolation procedure, which has relied thus far exclusively on liquid cultivation. While plating for contiguous lawns of Prochlorococcus has proven to be productive (15), attempts at colony formation (by pour plating or surface streak plating) have thus far met with significantly less success. Recovery efficiencies of the pour plating technique of 0.1 to 10% have been reported previously for some strains (15,24), but this technique has yet to produce pure cultures (15). The inability to readily obtain clonal, pure cultures of Prochlorococcus has severely limited progress in the genetic and physiological analysis of this ecologically important lineage.The "helper" phenotype of heterotrophic bacteria. Standard dilution streaking of contaminated Prochlorococcus cultures onto semisolid medium failed to produce axenic colonies. Colonies formed only within a visible mass of the contaminant heterotrophic bacteria; such masses appeared typically at the sites of the earliest, heaviest dilution streaks (data not shown). One interpretation of these results was that Prochlorococcus was able to grow only in the presence of the contaminating bacteria, perhaps because the bacteria provide a growth factor and/or remove an inhibitory factor. Coculturing with heterotrophic bacteria is required for the growth of some bacterial isolates (9) and is known to improve the growth of dinoflagellates (2, 7), suggesting that a similar interaction may help Prochlorococcus. To test this hypothesis, a heterotrophic contaminant (designated EZ55) of a culture of the ...

Transcriptional response of Prochlorococcus to co-culture with a marine Alteromonas: differences between strains and the involvement of putative infochemicals

“…Specifically, cysteine and methionine residues are especially sensitive to ROS (Arts et al, 2015). Although heterotrophic bacteria, including some Alteromonas strains, may scavenge ROS and thus reduce potential oxidative stress affecting Prochlorococcus (Morris et al, 2008;Morris et al, 2011), other marine heterotrophic bacteria, again including Alteromonads, in fact produce extracellular superoxide (Diaz et al, 2013). Additionally, the killing mechanism of many antibiotics ultimately involves the generation of ROS (reviewed by Dwyer et al, 2009).…”

“…Although significant advances have been made in understanding 'top down' control of Prochlorococcus populations by phage and grazers (for example, Worden and Binder, 2003;Avrani et al, 2011;Pasulka et al, 2015), as well as 'bottom up' control by nutrients, temperature and light (for example, Bouman et al, 2006;Johnson et al, 2006;Saito et al, 2014), studying how co-occurring heterotrophic microbes affect Prochlorococcus is still in its infancy. Several recent studies have shown that co-occurring heterotrophic bacteria can both enhance and inhibit the growth of Prochlorococcus in laboratory co-cultures (Morris et al, 2008;Sher et al, 2011). Furthermore, such interactions may have significant effects on the viability of Prochlorococcus in the oceans, for example through scavenging by heterotrophic bacteria of reactive oxygen species (ROS) produced by Prochlorococcus (Morris et al, 2011(Morris et al, , 2012.…”

Enrichment and characterization of ammonia-oxidizing archaea from the open ocean: phylogeny, physiology and stable isotope fractionation

“…N. yellowstonii and N. gargensis, the two cultivated thermophilic strains of AOA, also could not be established in pure culture (Hatzenpichler et al, 2008;de la Torre et al, 2008). This may be because there is a cooperative relationship between the bacteria and the archaea in these enrichment cultures, as has been reported in cultures of the marine chlorophyte Prochlorococcus (Morris et al, 2008), and low cell density dilutions that do not contain the associated heterotrophic bacteria are unable to grow. Interactions between the heterotrophic bacteria and autotrophic archaea in the enrichment, and in the ocean, will be an exciting area of future research.…”

Torn apart and reunited: impact of a heterotroph on the transcriptome of Prochlorococcus

“…Bulk chlorophyll fluorescence and flow cytometry samples were collected daily to monitor cell abundance; Prochlorococcus were counted based on chlorophyll fluorescence, and heterotrophs were enumerated following SYBR staining. The purity of axenic NATL2A cultures was verified before and after the experiment by testing for growth in three purity broths (ProAC, ProMM and MPTB (Saito et al, 2002;Morris et al, 2008;Berube et al, 2015)) and by flow cytometry. Transcriptome samples were collected by removing 10 ml of culture and placing it immediately into 30 ml of cold RNALater, then incubating these samples for 1-3 days at 4°C.…”

“…Growth of Prochlorococcus-the numerically dominant phytoplankter in the world's oceans (Flombaum et al, 2013)-in culture is improved by the presence of certain heterotrophs in terms of longevity, stationary phase cell density and ability to grow from low cell density (Sher et al, 2011). This phenomenon is attributable at least in part to the role of heterotrophs in reducing the levels of toxic reactive oxygen species such as hydrogen peroxide (Morris et al, 2008(Morris et al, , 2011. This is thought to compensate for the absence of catalase in the Prochlorococcus genome, indicating that interspecies interactions have clearly influenced the evolutionary selective pressures and processes acting upon this group of organisms (Morris et al, 2012).…”