Physiology and evolution of nitrate acquisition in Prochlorococcus

Abstract

Prochlorococcus is the numerically dominant phototroph in the oligotrophic subtropical ocean and carries out a significant fraction of marine primary productivity. Although field studies have provided evidence for nitrate uptake by Prochlorococcus, little is known about this trait because axenic cultures capable of growth on nitrate have not been available. Additionally, all previously sequenced genomes lacked the genes necessary for nitrate assimilation. Here we introduce three Prochlorococcus strains capable of growth on nitrate and analyze their physiology and genome architecture. We show that the growth of high-light (HL) adapted strains on nitrate is B17% slower than their growth on ammonium. By analyzing 41 Prochlorococcus genomes, we find that genes for nitrate assimilation have been gained multiple times during the evolution of this group, and can be found in at least three lineages. In low-light adapted strains, nitrate assimilation genes are located in the same genomic context as in marine Synechococcus. These genes are located elsewhere in HL adapted strains and may often exist as a stable genetic acquisition as suggested by the striking degree of similarity in the order, phylogeny and location of these genes in one HL adapted strain and a consensus assembly of environmental Prochlorococcus metagenome sequences. In another HL adapted strain, nitrate utilization genes may have been independently acquired as indicated by adjacent phage mobility elements; these genes are also duplicated with each copy detected in separate genomic islands. These results provide direct evidence for nitrate utilization by Prochlorococcus and illuminate the complex evolutionary history of this trait.

Global biogeography of Prochlorococcus genome diversity in the surface ocean

“…Additionally, the greater variability in the genomic context of environmental assemblies versus sequenced strains suggests that the assemblies are capturing new genomic backbones to some extent. Thus strains in culture may not fully represent Prochlorococcus diversity as it relates to temperature adaptation-as was recently observed for nitrogen assimilation (Martiny et al, 2009b;Berube et al, 2015). In Synechococcus, variation has been observed in the thermal niches of strains consistent with the thermal range of their isolation location.…”

“…After mapping to protein-coding regions using BLASTn, we used hmmalign from HMMER 3.1b1 (Eddy, 2011) to align sequences to the individual gene reference alignments of the corresponding orthologous group PPlacer (Matsen et al, 2010) coupled with RAxML (GTRGAMMA model) (Stamatakis, 2014) to generate the appropriate phylogenetic model statistics and map the sequences onto the reference phylogeny one core gene at a time, and finally we collapsed single gene branch abundance matrices yielding a phylogenetic distribution across samples (Supplementary Figure S2). Clades were defined by previous annotations (Berube et al, 2015) except HLII, which was split into clades with members o10% different (DNADIST, F84 model) and bootstrap values 475 (Felsenstein, 2005); deep branches were grouped as in Supplementary Figure S2. We excluded samples with o500 sequences and rarefied to account for sampling depth biases.…”

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

“…Prochlorococcus cultures were grown at 21°C under constant illumination (30 μmol photons m − 2 s − 1 ). A. macleodii MIT1002 (Biller et al, 2015b) was maintained at 21°C in ProMM medium (Pro99 media, as above, plus lactate, pyruvate, glycerol, acetate and Va vitamins) (Berube et al, 2015).…”

“…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.…”

Contribution of cyanobacterial alkane production to the ocean hydrocarbon cycle

“…Axenic cultures of Prochlorococcus CCMP1986 (MED4), MIT9312, and MIT9313 were used. Cultures were routinely assessed for purity by confirming a lack of turbidity after inoculation into three different purity test broths (53). Triplicate 2-L cultures of each strain were grown in Pro99 medium (54) prepared with 0.2 μm filtered, autoclaved seawater (collected from Vineyard Sound, MA) and supplemented with 10 mM filter-sterilized sodium bicarbonate upon inoculation.…”