Draft genomes of three closely related low light-adapted Prochlorococcus

Abstract

Objectives

The marine cyanobacterium Prochlorococcus is a critical part of warm ocean ecosystems and a model for studying microbial evolution and ecology. To expand the representation of this organism’s vast wild diversity in sequence collections, we performed a set of isolation efforts targeting low light-adapted Prochlorococcus. Three genomes resulting from this larger body of work are described here.

Data description

We present draft-quality Prochlorococcus genomes from enrichment cultures P1344, P1361, and P1363, sampled in the North Pacific. The genomes were built from Illumina paired reads assembled de novo. Supporting datasets of raw reads, assessments, and sequences from co-enriched heterotrophic marine bacteria are also provided. These three genomes represent members of the low light-adapted LLIV Prochlorococcus clade that are closely related, with 99.9% average nucleotide identity between pairs, yet vary in gene content. Expanding the powerful toolkit of Prochlorococcus genomes, these sequences provide an opportunity to study fine-scale variation and microevolutionary processes.

Objective

Inhabiting the oligotrophic open oceans, the cyanobacterium Prochlorococcus is the most abundant photoautotroph on Earth, influencing local trophic flow and global biogeochemical cycling [1, 2]. A minimalist adapted to its low-nutrient environment [3, 4], Prochlorococcus is unique among cyanobacteria for its small cells, 0.5 – 1.0 µm diameter [5, 6], and small genomes, 1.6 – 2.7 Mb [7]. As a model system, Prochlorococcus has revealed ecological structure and adaptation at many scales (e.g., [7,8,9,10,11,12]).

Prochlorococcus diversity is organized into phylogenetic clades, sorted into low light-adapted (LL) and high light-adapted (HL) groups based on ecological, genomic, and physiological patterns [7, 12, 13]. While HL clades are more abundant [9, 14], LL Prochlorococcus include lineages adapted to uniquely challenging conditions, including anoxic zones [15], seasonal mixing [14], and light limitation in the deep euphotic zone [12]. LL strains have larger genomes with substantial flexible genome content [7]. Known wild diversity among LL Prochlorococcus far exceeds culture collections [7], and culturing this organism can be unpredictable [16].

In this context, we performed isolation and sequencing efforts targeting LL Prochlorococcus during a North Pacific sampling opportunity. This work resulted in previously described strains [17,18,19] and the three LLIV clade genomes described here: P1344, P1361, and P1363. To date, these new genomes have been used in studies on novel mobile genetic elements [20] and diversity in Prochlorococcus enrichment cultures [19]. These genomes represent a set of sympatric, closely related strains, supporting the study of microevolution in the growing Prochlorococcus sequence collection.

Data description

We present draft Prochlorococcus genomes from enrichment cultures P1344, P1361, and P1363 (Dataset 1) [21,22,23]. They come from a single sample collected in the North Pacific from 150 m at Station ALOHA (22.75°N, 158°W), June 2013 [24, 25]. Isolation protocols [16] were tuned to enrich for LL Prochlorococcus. Data file 1 [26] provides detailed methods; Table 1 lists datasets. Previously described strains from the same project [17,18,19] are described in Data file 2 [26]. After 1.5 years of subculturing, enrichments P1344, P1361, and P1363 each stabilized to a single internal transcribed spacer rRNA (ITS) sequence [27], an indicator of unialgal Prochlorococcus cultures [16, 28]. Because the time from sea to genome was shorter than for previously sequenced enrichment cultures (e.g., 5–20 years [28]), we followed a naming convention for Prochlorococcus enrichments [29]. The three ITS sequences, in the LLIV clade, were identical to each other, strains MIT1312 and MIT1327 (additional co-isolates from the same sample [17]), and MIT1227 (from Station ALOHA one year earlier [30]).

Table 1 Overview of data files/data sets

Genomic libraries were prepared as in [34] from bulk enrichment DNA and sequenced with Illumina MiSeq V3 at the MIT BioMicroCenter [35] with 300 base paired reads. Raw reads are available in the NCBI Sequencing Read Archive (Dataset 2) [31,32,33]. Quality-trimmed reads were assembled de novo with SPAdes v.3.1.1 [36]. Enrichment contigs were screened with blastn [37] against the NCBI nt database to separate out Prochlorococcus sequences (Data file 3) [26]. Contigs with at least 500 bases, top BLAST hits to Prochlorococcus (Data file 4) [26], and at least 2 × kmer coverage were selected to produce the genomes. For P1344, P1361, and P1363, respectively, genomes consist of 106, 45, and 66 contigs, with average read coverage depths 82x, 57x, and 67x [38] and genome sizes 2.47 Mb, 2.51 Mb, and 2.56 Mb, similar to other LLIV genomes (Data file 2) [26].

For initial assessments, we annotated the genomes with Prokka [39] (Dataset 3, Data file 5) [26]. Genbank annotations come from the NCBI automated annotation pipeline (Dataset 1) [22,23,24]. Enrichment assembly (Dataset 4 [26]) BLAST and metagenomic binning results (Data files 1, 3, 6, 7) [26] show the presence of copiotrophic marine bacteria at lower coverage than Prochlorococcus, with partially recovered genomes [40,41,42,43]. These include Alteromonas and Marinobacter, groups previously studied in co-culture with Prochlorococcus that can enhance its growth or survival in culture [18, 19, 44,45,46]. Comparisons among P1344, P1361, P1363, and the three other genomes with the same ITS (Data files 1, 8, 9) [26] support the idea that they represent similar but distinct strains, with average nucleotide identity from 99.9% ± 0.7% to 100.0% ± 0.1% s.d. [47], 103 – 3,854 SNPs in pairwise alignments [48, 49], and 32 – 132 distinct genes in pairwise ortholog group comparisons [50]. While mostly without predicted functions, these variable genes include a pilus-related protein and a member of the cytochrome c family, located near contig ends (Data files 1, 5) [26]. This scale of variation will support the study of recent Prochlorococcus evolution.

Limitations

The enrichment cultures described here were lost during maintenance and are no longer available. These genomes came from enrichments rather than clones, representing a snapshot in time of a likely single dominant strain in each culture and an associated heterotrophic community (Data file 1) [26]. That these genomes are not linked to existing cultures and did not come from clonal isolates limits downstream use and requires caution in interpretation, but not more so than sequences derived from single cell methods or metagenomics, and with the benefit of more complete genomes. With care, these new genomes still have the potential to contribute useful insights on the nature and mechanisms of fine-scale evolution in Prochlorococcus.