Widespread metabolic potential for nitrite and nitrate assimilation among Prochlorococcus ecotypes

Abstract

The marine cyanobacterium Prochlorococcus is the most abundant photosynthetic organism in oligotrophic regions of the oceans. The inability to assimilate nitrate is considered an important factor underlying the distribution of Prochlorococcus, and thought to explain, in part, low abundance of Prochlorococcus in coastal, temperate, and upwelling zones. Here, we describe the widespread occurrence of a genomic island containing nitrite and nitrate assimilation genes in uncultured Prochlorococcus cells from marine surface waters. These genes are characterized by low GC content, form a separate phylogenetic clade most closely related to marine Synechococcus, and are located in a different genomic region compared with an orthologous cluster found in marine Synechococcus strains. This sequence distinction suggests that these genes were not transferred recently from Synechococcus. We demonstrate that the nitrogen assimilation genes encode functional proteins and are expressed in the ocean. Also, we find that their relative occurrence is higher in the Caribbean Sea and Indian Ocean compared with the Sargasso Sea and Eastern Pacific Ocean, which may be related to the nitrogen availability in each region. Our data suggest that the ability to assimilate nitrite and nitrate is associated with microdiverse lineages within high- and low-light (LL) adapted Prochlorococcus ecotypes. It challenges 2 long-held assumptions that (i) Prochlorococcus cannot assimilate nitrate, and (ii) only LL adapted ecotypes can use nitrite. The potential for previously unrecognized productivity by Prochlorococcus in the presence of oxidized nitrogen species has implications for understanding the biogeography of Prochlorococcus and its role in the oceanic carbon and nitrogen cycles.

Fig. 1.

Nitrogen acquisition genes in Prochlorococcus and Synechococccus. (A) Nitrogen genes in the genomic region between pyrG and ppk. (B) Suggested consensus sequence of nitrogen acquisition genes acquired in an uncultured lineage of Prochlorococcus and orthologous regions in HL Prochlorococcus. Green represents genes associated with nitrite assimilation, red represents genes associated nitrate assimilation, blue represents genes associated with urea assimilation, black represents other genes conserved across Prochlorococcus and Synechococcus, yellow represents conserved genes in Prochlorococcus, and the rest are gray. The red star represents genes that have paired end mates matching the region around the nitrate genomic island. It is important to note that all genes in this insertion region were detected on DNA fragments that also contained genes matching Prochlorococcus (i.e., the paired end sequence). We only used a scaffold to estimate the gene order. Note also that urea assimilation genes are located in a different genomic region in many Prochlorococcus strains.

Fig. 2.

Phylogeny of putative nitrite and nitrate assimilation protein sequences inferred from GOS expedition samples and Cyanobacteria strains. Next to the sequence ID is listed the organisms and locus most similar to the paired end sequence mate. (A) Nitrite reductase (NirA) (from position 221 to 513 in Synechococcus WH8102). (B) Conserved hypothetical protein (NirX) (all positions). (C) Nitrate reductase (NarB) (from position 1 to 280 in Synechococcus WH8102). (D) Molybdopterin biosynthesis protein A (MoaA) (from position 10 to 175 in Synechococcus WH8102). The phylogenetic trees are based on protein sequence similarity using neighbor-joining. Bootstrap values (total 100) are calculated using neighbor-joining, maximum parsimony, and maximum likelihood. Sequence ID refers to sample number and JCVI_READ_XXX. The best hit to the paired end mate of each sequence is found by blastx searching against all sequenced genomes in GenBank as of 01/28/08. Additional regions for each protein are detailed in the supplemental figures.

Fig. 3.

Distribution of Prochlorococcus and Synechococcus and associated nitrite and nitrate assimilation genes across ocean regions. (A) Distribution of Synechococcus, LL, and HL Prochlorococcus based the average abundance of 8 single copy core genes. (B) Distribution of high and low GC copies of genes orthologous to nitrite and nitrate assimilation genes in Synechococcus. Abundance is normalized according to gene length.