Ecological and functional capabilities of an uncultured Kordia sp.

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Abstract

Cultivable bacteria represent only a fraction of the diversity in microbial communities. However, the official procedures for classification and characterization of a novel prokaryotic species still rely on isolates. Nevertheless, due to single cell genomics, it is possible to retrieve genomes from environmental samples by sequencing them individually, and to assign specific genes to a specific taxon, regardless of their ability to grow in culture. In this study, a complete description was performed for uncultured Kordia sp. TARA_039_SRF, a proposed novel species within the genus Kordia, using culture-independent techniques. The type material was a high-quality draft genome (94.97% complete, 4.65% gene redundancy) co-assembled using ten nearly identical single amplified genomes (SAGs) from surface seawater in the North Indian Ocean during the Tara Oceans Expedition. The assembly process was optimized to obtain the best possible assembly metrics and a less fragmented genome. The closest relative of the species was Kordia periserrulae, which shared 97.56% similarity of the 16S rRNA gene, 75% orthologs and 89.13% average nucleotide identity. The functional potential of the proposed novel species included proteorhodopsin, the ability to incorporate nitrate, cytochrome oxidases with high affinity for oxygen, and CAZymes that were unique features within the genus. Its abundance at different depths and size fractions was also evaluated together with its functional annotation, revealing that its putative ecological niche could be particles of phytoplanktonic origin. It could putatively attach to these particles and consume them while sinking to the deeper and oxygen depleted layers of the North Indian Ocean.

Introduction

There is a lack of consensus on which ecologically coherent units should be used as a proxy for bacterial species in environmental communities [23], [52], [67], [72]. Moreover, for a new bacterial species to be officially recognized, its isolation in pure culture is still a requirement. Therefore, it becomes necessary to re-define bacterial “species” to fit the reality of the (still uncultured) majority of bacteria. An update of the validation of novel uncultured high-quality genomes is also needed, as they are given a provisional Candidatus status even after a thorough description [37]. Recent efforts have emerged to facilitate the standardization of novel uncultured taxa [37], such as the Microbial Genome Atlas (MiGA) that infers genome-based taxonomy and quality assessment across genomes from different environments [66].

Nowadays, uncultured genomes can be used to expand the knowledge of the genetic and evolutionary differences between representatives of the same species or close relatives, as well as to enrich the knowledge of microbial diversity. The comparison between MAGs and SAGs assembled from the same Baltic Sea water samples, has revealed very high nucleotide identities between the corresponding pairs but a difference in the size and completeness between the genomes [4]. Uncultured genomes can be retrieved by: (i) co-assembling metagenomes (metagenomic assembled genomes; MAGs), or (ii) single cell genomics (single amplified genomes; SAGs). In marine microbiology, assembling MAGs has provided a vast number of genomes that belong to novel bacterial and archaeal phyla, unveiling key players in the biogeochemistry of the oceans [18], [56], [57]. Alternatively, single cell genomics allows the assignment of specific functional traits to a specific taxon, which is an outstanding resolution level for microdiversity studies. Single cell genomics has helped link functional roles to relevant taxonomic groups that have changed the current understanding of predominant marine metabolisms (such as chemolithoautotrophy or the role of nitrite-oxidizing bacteria in the deep ocean) [53], [77]. While MAGs tend to be longer and more complete than SAGs, MAGs result from a population of genomes and there is still lack of consensus about what taxonomic units are they reflecting. Nevertheless, multiple SAGs can be co-assembled to retrieve more complete genomes, provided they are closely related phylogenetically. SAGs have also been used in pan-genome analyses, which had previously been restricted mostly to cultured microorganisms (especially pathogenic bacteria) [49], [51], [79]. Comparative genomics and the development of the “pan-genome” concept offers an alternative for understanding the genetic extent and dynamics of bacterial species [49], [51], [79], dramatically changing the description of a species by studying multiple genomes belonging to the same defined taxa [34], [38], [65]. In the marine environment, SAG-based pan-genome analyses have focused mostly on the highly studied Prochlorococcus genus (revealing hundreds of co-existing Prochlorococcus populations [31] and the link between their hypervariable genomic islands and the environment [17]) or together with SAR11 (defining endemic gene-level adaptations to specific locations such as the Red Sea [80]).

In the present study, the co-assembly of ten SAGs was undertaken from a nearly clonal population of a novel species in the genus Kordia in order to generate a high-quality genome, complete enough to: (i) allow its putative functional description, (ii) determine its preferred niche in the water column from which it was retrieved, and (iii) to describe the novel species in comparison with the available sequenced relatives of the genus Kordia. The genus Kordia belongs to the family Flavobacteriaceae and it was first proposed with the isolation in culture of Kordia algicida, which lyses algal cells and feeds on phytoplanktonic bloom exudates [75]. Members of this genus are Gram-negative, strictly aerobic or facultatively anaerobic, non-motile or motile by gliding, rod shaped, and show a 34–37% DNA G + C content [33]. In the last 15 years, new species have been added to the genus, and all of them have been isolated from samples found in the aquatic environment: K. zhangzhouensis thrives in freshwater [41], K. jejudonensis was isolated from the interphase between seawater and freshwater springs [54], K. antarctica and K. aquimaris were isolated from Antarctic and Taiwanese seawater, respectively [6], [26], K. ulvae, K. zosterae and Kordia sp. SMS9 were retrieved from the surface of marine algae [33], [59], [62], and K. periserrulae was found in the gut of a tidal flat polychaete [14]. Kordia sp. NORP58 is a MAG assembled from a marine subsurface aquifer [81]. At the time of writing, the genus consists of eight species with validly published names, of which four have had their genomes completely sequenced.

Despite the fundamental insights into microbial ecology and evolution provided by the analyses of uncultured bacterial and archaeal genomes, there is still no proper taxonomy for the uncultured majority. Therefore, this study aimed to provide a good example of a complete description of a novel species of the genus Kordia analyzed via culture-independent methods in order to infer its ecological and functional description.

Section snippets

Single-amplified genomes generation and phylogenetic analysis

A total of 98 single-amplified genomes were generated as detailed in Ref. [77] from a surface (SRF) seawater sample from the North Indian Ocean (latitude 18.59 °N – longitude 66.62 °E) during the circumnavigation expedition Tara Oceans [30], station TARA_039 (Sample ID: TARA_G000000266) (Supplementary Table S1).

Multilocus sequence analysis (MLSA) of the generated SAGs revealed that 84% of them belonged to a novel species of the genus Kordia (referred to hereafter as Kordia sp. TARA_039_SRF) and

Co-assembly of the Kordia sp. TARA_039_SRF genome

Ten SAGs belonging to the genus Kordia, provisionally named Kordia sp. TARA_039_SRF, were selected for genome co-assembly and description of gene content. These SAGs were tested for microdiversity through MLSA analyses, which resulted in 100% identity at the nucleotide level in each marker [69]. The five markers were the 16S rRNA gene, partial 23S rRNA gene and the internal transcribed spacer, which could be amplified in the ten SAGs, the partial RNA polymerase subunit B, amplified in five

Central metabolism

The co-assembled Kordia sp. TARA_039_SRF had the ability to shut down the loss of CO2 in the regular TCA cycle by the glyoxylate shunt, like other Kordia spp., and it was also able to replenish the TCA cycle with certain intermediates through anaplerotic routes: it encoded both PEP carboxykinase and PEP carboxylase, which convert PEP to oxaloacetate using ATP and CO2, and H2O and HCO3, respectively. This mechanism is also found in other Kordia spp. Bicarbonate uptake can be achieved through

Carbohydrate-active enzymes, PULs, surface adhesion and motility

Kordia sp. TARA_039_SRF encoded a CAZymes array most similar to K. periserrulae and larger than the other Kordia spp. included in the study (Supplementary Table S6). It showed the second highest density of carbohydrate-active enzymes (41 enzymes per genomic Mbp) after that of K. zhangzhouensis. It encoded 34 non-catalytic carbohydrate-binding modules (CBM), mostly specific for complex carbohydrates, such as cellulose, chitin, mannan, beta-glucans, starch, and glycogen. The novel species also

Sulfur and nitrogen metabolism

Another unique feature of Kordia sp. TARA_039_SRF within its genus was that it coded for all the genes involved in the assimilatory sulfate reduction pathway that converts sulfate to sulfide, and it was also able to transform thiosulfate to sulfite.

The genome of Kordia sp. TARA_039_SRF was the only Kordia genome sequenced to date that encoded both NasA, a nitrate/nitrite transporter, and NasF, the substrate binding protein in the nitrate/nitrite transport system. The bacterium was predicted to

Light sensing and environmental information processing

The co-assembled Kordia encoded a copy of a green-light absorbing proteorhodopsin, the same as K. periserrulae and Kordia sp. SMS9. Their phylogenetic reconstruction clustered the proteins of the co-assembly and K. periserrulae in the same clade with a 100% bootstrap value (Supplementary Fig. S3), and their pairwise alignment showed that they were identical in 95.5% of the sites (232 of 243 bp).

It also coded for other putative light sensors usually found in proteorhodopsin-containing marine

Transporters

The co-assembly encoded 95 different transporter families and ∼88 transporters per Mbp. A total of four different types of pore-forming toxins were found, together with phage-related transporters, such as the FadL outer membrane protein and a holin belonging to the Bacillus subtilis phage φ29 transporter family, two different light-absorption driven transporters and uptake systems specific for amino acids, potassium, fatty acids, nitrate, dissolved inorganic carbon, iron and magnesium (Fig. 3;

Pigment and vitamin synthesis

Kordia sp. TARA_039_SRF was the only described Kordia spp. that could potentially synthesize beta-carotene without relying on any exogenous intermediates, since it coded for the complete terpenoid backbone synthesis. Another exclusive feature was the putative ability to synthesize biotin from malonyl-ACP and pimeloyl-CoA. As other Kordia spp., Kordia sp. TARA_039_SRF could synthesize riboflavin (vitamin B2) and it also had genes related to the synthesis of folate (B9), pantothenate (B5) and

Genomic islands, mobile elements and prophages

Kordia sp. TARA_039_SRF’s genome contained 12 predicted genomic islands with a total length of 252,407 bp (5.5% of the total genome and 28% of the unique accessory genome). They coded for virulence genes such as non-ribosomal peptides (NRPs), porins, toxins and invasion proteins, and other genes such as those involved in oxidative phosphorylation, nitrate/nitrite transport, and sulfur carrier proteins (Supplementary Table S12). There were 16 different types of transporters encoded in ten genomic

Discussion

The aim of this study was to characterize the novel uncultured species Kordia sp. TARA_039_SRF and shed light on its ecological and functional potential in ocean waters through an optimized co-assembly of ten co-occurring and nearly identical marine Kordia SAGs.

A total of ten Kordia SAGs previously classified as nearly identical genomes [69] were co-assembled into a high-quality draft genome, which was 94.83% complete and met the standards set regarding contamination (<5%) [11], [37]. Recent

Author’s contributions

SA designed this research. MR-L performed the laboratory and analyses work and wrote the paper. PS and JG helped with the data analyses. SA funded this research with contributions from CP-A and JG. All authors were involved in critical reading for writing the paper.

Acknowledgements

High-Performance computing analyses were run at the Marine Bioinformatics Core Facility (MARBITS) of the Institut de Ciències del Mar (ICM-CSIC) in Barcelona. We thank Shook Studio for assistance with figure design and execution. We thank Aharon Oren for helping in the etymology of the candidatus and Dr. Ramon Roselló-Móra and the two other anonymous reviewers for helping us improve our manuscript. We thank our fellow scientists, the crew and chief scientists of the different cruise legs

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