1 2 3 4 5 6 7 Ecological and functional capabilities of an uncultured 8 Kordia sp. 9 10 Royo-Llonch M1,4, Sánchez P1, González JM2, Pedrós-Alió C3, Acinas SG1* 11 12 _____________________________________________________________________ 13 14 1 Department oF Marine Biology and Oceanography, Institut de Ciències del Mar (ICM), 15 CSIC, Barcelona, Spain. 16 2 Department oF Microbiology, University oF La Laguna, La Laguna, Spain 17 3 Systems Biology Program, Centro Nacional de Biotecnología (CNB), CSIC, Madrid, Spain 18 4 Departament de Genètica i Microbiologia, Facultat de Biociències, Universitat Autònoma 19 de Barcelona, Bellaterra, Spain 20 21 *Correspondence: 22 Dr. Silvia G. Acinas 23 [email protected]; 24 25 Keywords: SAG , Kordia, co-assembly, photoheterotrophy, metagenomics 26 1 27 Abstract 28 Cultivable bacteria are only a Fraction oF the diversity in microbial communities. However, 29 the oFFicial procedures For classiFication and characterization of a novel prokaryotic 30 species still rely on isolates. Thanks to Single Cell Genomics, it is possible to retrieve 31 genomes From environmental samples by sequencing them individually, and to assign 32 speciFic genes to a speciFic taxon, regardless oF their ability to grow in culture. In this 33 study, we perFormed a complete description oF the uncultured Kordia sp. 34 TARA_039_SRF, a proposed novel species within the genus Kordia, using culture- 35 independent techniques. The type material was a high-quality draFt genome (94.97% 36 complete, 4.65% gene redundancy) co-assembled using 10 nearly identical Single 37 AmpliFied Genomes (SAGs) From surFace seawater in the North Indian Ocean from the 38 Tara Oceans expedition. The assembly process was optimized to obtain the best 39 possible assembly metrics and a less Fragmented genome. Its closest relative is Kordia 40 periserrulae, sharing 97.56% similarity of the 16S rRNA gene, 75% of their orthologs and 41 89.13% average nucleotide identity. We describe the Functional potential oF the proposed 42 novel species, that includes proteorhodopsin, the ability to incorporate nitrate, 43 cytochrome oxidases with high aFFinity for oxygen and CAZymes that are unique features 44 within the genus. Its abundance at diFFerent depths and size Fractions was also evaluated 45 together with its Functional annotation, revealing that its putative ecological niche seems 46 to be particles oF phytoplanktonic origin. They can attach to these particles and consume 47 them while sinking to the deeper and oxygen depleted layers of the North Indian Ocean. 48 49 2 50 Introduction 51 There is a lack oF a consensus on what ecologically coherent units should be used as a 52 proxy For bacterial species in environmental communities [23,52,67,72]. Moreover, For a 53 new bacterial species to be oFFicially recognized, its isolation in pure culture is still a 54 requirement. It becomes necessary to re-define bacterial “species” to fit with the reality 55 of the (still uncultured) majority oF bacteria. An update of the validation oF novel 56 uncultured high-quality genomes is also needed, as they are given a provisional 57 Candidatus status even after a thorough description [37]. Recent eFForts have emerged 58 to Facilitate novel uncultured taxa standardization [37], like the Microbial Genome Atlas 59 (MiGA), which inFers genome-based taxonomy and quality assessment across genomes 60 From diFFerent environments [66]. 61 Nowadays, uncultured genomes can be used to expand knowledge oF the genetic and 62 evolutionary diFFerences between representatives oF the same species or close relatives, 63 as well as For enriching knowledge oF microbial diversity. Uncultured genomes can be 64 retrieved by: i) co-assembling metagenomes (Metagenomic Assembled Genomes or 65 MAGs) or ii) by single cell genomics (Single AmpliFied Genomes or SAGs). In marine 66 microbiology, assembling MAGs has brought a vast amount oF genomes belonging to 67 novel bacterial and archaeal phyla, unveiling key players in the biogeochemistry oF the 68 oceans [18,56,57]. Alternatively, Single Cell Genomics allows the assignment oF speciFic 69 Functional traits to a speciFic taxon, an outstanding resolution level For microdiversity 70 studies. Single Cell Genomics has helped link Functional roles to relevant taxonomic 71 groups that have changed the current understanding oF predominant marine 72 metabolisms (such as chemolithoautotrophy or the role oF nitrite-oxidizing bacteria in the 73 deep ocean) [53,77]. While MAGs tend to be longer and more complete than SAGs, 74 MAGs result From a population oF genomes and there is still lack oF consensus about 75 what taxonomic units are they reFlecting. Nevertheless, multiple SAGs can be co- 76 assembled to retrieve more complete genomes, provided they are closely related 3 77 phylogenetically. The comparison between MAGs and SAGs assembled From the same 78 Baltic Sea water samples, revealed very high nucleotide identities between the 79 corresponding pairs but a diFFerence in the size and completeness between the genomes 80 [4]. SAGs have also been used in pangenome analyses, which had previously been 81 mostly restricted to cultured microorganisms (especially pathogenic bacteria) [49,51,79]. 82 Comparative genomics and the development oF the “pan-genome” concept oFFered an 83 alternative For understanding the genetic extent and dynamics oF bacterial species 84 [49,51,79], dramatically changing the description oF a species by studying multiple 85 genomes belonging to the same deFined taxa [34,38,65]. In the marine environment, 86 SAG-based pangenome analyses have mostly Focused on the highly studied 87 Prochlorococcus (revealing hundreds oF co-existing Prochlorococcus populations [31] 88 and the link between their hypervariable genomic islands and the environment [17]) or 89 together with SAR11 (deFining endemic gene-level adaptations to speciFic locations like 90 the Red sea [80]). 91 In the present study, we carried out the co-assembly oF 10 SAGs oF a nearly clonal 92 population oF a novel species in the genus Kordia to generate a high-quality genome 93 complete enough to: i) allow its putative Functional description, ii) determine its preFerred 94 niche in the water column From which it was retrieved, and iii) to describe the novel 95 species in comparison with the available sequenced relatives oF the genus Kordia. The 96 genus Kordia belongs to the Family Flavobacteriaceae and it was First proposed with the 97 isolation in culture oF Kordia algicida, which lyses algal cells and Feeds on 98 phytoplanktonic bloom exudates [75]. Members oF this genus are Gram-negative, strictly 99 aerobic or Facultatively anaerobic, non-motile or motile by gliding, rod shaped and 100 showing 34-37% of DNA G+C content [33]. In the last 15 years, new species have been 101 added to the genus, all oF them isolated From samples Found in the aquatic environment: 102 K. zhangzhouensis thrives in Freshwater [41], K. jejudonensis was isolated From the 103 interphase between seawater and Freshwater springs [54], K. antarctica and K. aquimaris 4 104 were isolated From Antarctic and Taiwanese seawater, respectively [6,26], K. ulvae, 105 K.zosterae and Kordia sp. SMS9 were retrieved from the surFace oF marine algae 106 [33,59,62] and K. periserrulae was Found in the gut oF a tidal Flat polychaete [14]. Kordia 107 sp. NORP58 is a MAG assembled From a marine subsurFace aquiFer [81]. At the time oF 108 writing, the genus consists oF eight species with validly published names, oF which four 109 have had their genomes completely sequenced. 110 111 Despite the Fundamental insights in microbial ecology and evolution provided by the 112 analyses oF uncultured bacterial and archaeal genomes, there is not yet a proper 113 taxonomy For the uncultured majority. We aim to provide a good example oF a complete 114 description oF a novel species of the genus Kordia analyzed via culture-independent 115 methods to inFer its ecological and Functional description. 116 5 117 Materials and Methods 118 Single AmpliFied Genome generation and phylogeny analysis 119 A total oF 98 Single AmpliFied Genomes were generated as detailed in [77] from a surFace 120 (SRF) seawater sample From the North Indian Ocean (latitude 18.59ºN – longitude 121 66.62ºE) during the circumnavigation expedition Tara Oceans [30], station TARA_039 122 (Sample ID: TARA_G000000266)(SM Table 1). 123 Multi Locus Sequencing Analysis (MLSA) oF the generated SAGs revealed that 84% oF 124 them belonged to a novel species oF the genus Kordia (reFerred hereaFter as Kordia sp. 125 TARA_039_SRF) and that the ampliFied genes (16S rRNA, partial 23S rRNA, partial 126 RNA polymerase subunit B and partial proteorhodopsin genes, as well as Internally 127 Transcribed Spacer) were identical in the whole Kordia SAG population. More details on 128 the phylogeny and distribution oF these Kordia SAGs can be Found in [69]. 129 SAG selection 130 We chose 10 out oF the 98 generated Kordia SAGs For Whole Genome Sequencing. 131 They were selected based on: i) Multiple Displacement AmpliFication’s (MDA) Cp values, 132 since they were the shortest, ranging from 7:15-8:15, and ii) that ampliFication was 133 possible For any oF the markers tested in previous MLSA. 134 Sequencing and read treatment 135 Sequencing oF the ten SAGs was carried out in two batches. For the First set oF Five, the 136 sequence reads were obtained by Illumina MiSeq 2x300 bp technology and by Illumina 137 HiSeq 2x250 bp For the second set. Quality assessment oF the raw reads was perFormed 138 using FastQC and Illumina PhiX174 adapters were removed using Bowtie2 v2.2.9 [43] 139 and Samtools v.1.3.1 [46]. AFterwards, reads were normalized by coverage For each 140 individual SAG using DOe JGI’s BBnorm, setting the maximum coverage at 40X. The 141 normalized paired-end libraries were trimmed and Filtered with Trimmomatic [10] and 142 paired-reads were merged with PeAR v0.9.6 [85].