bioRxiv preprint doi: https://doi.org/10.1101/2020.11.18.388454; this version posted November 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Characterization of the first cultured free-living representative of 2 Candidatus Izimaplasma uncovers its unique biology 3 Rikuan Zheng1,2,3,4, Rui Liu1,2,4, Yeqi Shan1,2,3,4, Ruining Cai1,2,3,4, Ge Liu1,2,4, Chaomin Sun1,2,4* 1 4 CAS Key Laboratory of Experimental Marine Biology & Center of Deep Sea 5 Research, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China 2 6 Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory 7 for Marine Science and Technology, Qingdao, China 3 8 College of Earth Science, University of Chinese Academy of Sciences, Beijing, 9 China 10 4Center of Ocean Mega-Science, Chinese Academy of Sciences, Qingdao, China 11 12 * Corresponding author 13 Chaomin Sun Tel.: +86 532 82898857; fax: +86 532 82898857. 14 E-mail address: [email protected] 15 16 17 Key words: Candidatus Izimaplasma, uncultivation, biogeochemical cycling, 18 extracellular DNA, in situ, deep sea 19 Running title: Characterization of the first cultured Izimaplasma 20 21 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.18.388454; this version posted November 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 22 Abstract 23 Candidatus Izimaplasma, an intermediate in the reductive evolution from Firmicutes 24 to Mollicutes, was proposed to represent a novel class of free-living wall-less bacteria 25 within the phylum Tenericutes found in deep-sea methane seeps. Unfortunately, the 26 paucity of marine isolates currently available has limited further insights into their 27 physiological and metabolic features as well as ecological roles. Here, we present a 28 detailed description of the phenotypic traits, genomic data and central metabolisms 29 tested in both laboratorial and deep-sea environments of the novel strain zrk13, which 30 allows for the first time the reconstruction of the metabolic potential and lifestyle of a 31 member of the tentatively defined Candidatus Izimaplasma. On the basis of the 32 description of strain zrk13, the novel species and genus Xianfuyuplasma coldseepsis is 33 proposed. Notably, DNA degradation driven by X. coldseepsis zrk13 was detected in 34 both laboratorial and in situ conditions, strongly indicating it is indeed a key DNA 35 degrader. Moreover, the putative genes determining degradation broadly distribute in 36 the genomes of other Izimaplasma members. Given extracellular DNA is a 37 particularly crucial phosphorus as well as nitrogen and carbon source for 38 microorganisms in the seafloor, Izimaplasma bacteria are thought to be important 39 contributors to the biogeochemical cycling in the deep ocean. 40 41 42 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.18.388454; this version posted November 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 43 Introduction 44 The phylum Tenericutes is composed of bacteria lacking a peptidoglycan cell wall 45 and consists of bacteria that evolved from the phylum Firmicutes1-3. Two distinctive 46 features set Tenericutes apart from the Firmicutes: the inability to synthetize 47 precursors of peptidoglycan and thereby forming a cell wall4-7, and extreme reduction 48 of the size of genome (between 530 to 2220 kbp)8,9. Tenericutes includes the class 49 Mollicutes2, three taxa in provisional class Candidatus Izimaplasma4, and several taxa 50 of unclassified status. Tenericutes bacteria have evolved a broad range of lifestyles, 51 including free-living, commensalism and parasitism2. Up to date, almost all reported 52 Mollicutes (including five orders Mycoplasmatales, Entomoplasmatales, 53 Haloplasmatales, Acholeplasmatales, and Anaeroplasmatales)10 are commensals or 54 obligate parasites of humans, domestic animals, plants and insects2. In comparison, 55 some free-living species are found to be associated with inert substrates (e.g. 56 Candidatus Izimaplasma)4 or animal/plant surfaces (e.g. Acholeplasma laidlawii)11,12. 57 Tenericutes bacteria ubiquitously exist in numerous environments13. 58 Environmental 16S rRNA surveys have identified a large number of unknown 59 Tenericutes clades in diverse environments including the deep oceans, introducing the 60 possibility that these Tenericutes may represent free-living microorganisms which 61 conducting a non-host-associated life style2. Indeed, free-living Candidatus 62 Izimaplasma4 and Haloplasma14 were reported in a deep-sea cold seep and brine pool, 63 respectively. These deep-sea free-living Tenericutes exhibit metabolic versatility and 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.18.388454; this version posted November 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 64 adaptive flexibility, showing the possibility to isolate more Tenericutes bacteria from 65 the oceans and even other extreme environments. 66 Among the free-living Tenericutes identified in the marine environments, 67 Candidatus Izimaplasma was mainly studied based on the enrichment and 68 corresponding assembled genomes given the absence of pure cultures4,15. Candidatus 69 Izimaplasma was proposed to represent a novel class of free-living wall-less bacteria 70 within the phylum Tenericutes4, and an intermediate in the reductive evolution from 71 Firmicutes to Mollicutes4. Members of the Izimaplasma are thought to be heterotrophs 72 engaging primarily in fermentative metabolism4, and thereby producing lactate and 73 possibly other small molecules such as acetate or ethanol, which in turn may be 74 utilized by other members of the cold seep15. Notably, based on their genomic 75 analysis, Candidatus Izimaplasma bacteria are believed to be important DNA 76 degraders4,15. Recent studies have shown high concentrations of extracellular DNA in 77 marine sediments from shallow depths down to the abyssal floor16-18. Given 78 extracellular DNA is a particularly crucial phosphorus as well as nitrogen and carbon 79 source for microorganisms in the seafloor, Candidatus Izimaplasma might be a key 80 contributor to the biogeochemical cycling in the deep ocean. However, the paucity of 81 isolates currently available has greatly limited further insights into the physiological, 82 ecological and evolutional studies of Candidatus Izimaplasma. 83 In this study, we successfully cultivated the first free-living representative of the 84 class Candidatus Izimaplasma. Using growth assay and transcriptomic methods, we 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.18.388454; this version posted November 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 85 further found organic nutrient and thiosulfate could significantly promote the growth 86 of the new isolate. Of note, the novel isolate was found to be an effective degrader of 87 extracellular DNA in both laboratorial and real deep-sea conditions. Lastly, its 88 metabolic pathways were reconstructed based on multi-omics results presented in this 89 study and its potential ecological roles were also discussed. 90 Results 91 Quantification of the abundance of Tenericutes and Candidatus Izimaplasma in 92 the deep-sea cold seep sediments. To gain some preliminary understanding of 93 Tenericutes in the deep-sea, operational taxonomic units (OTUs) sequencing was 94 performed to detect the abundance of Tenericutes present in the cold seep sediments 95 at depth intervals of 0-10 cm, 30-50 cm, 90-110 cm, 150-170 cm, 210-230 cm, 96 230-250 cm from the surface to the deep layer. As previously reported2, the phylum 97 Tenericutes had a very low abundance in the cold seep sediments (Supplementary Fig. 98 1a). For example, the percentages of Tenericutes only respectively accounted for 99 0.011%, 0.222% and 0.003% of the whole bacterial domain in the samples ZC1, ZC3 100 and ZC4 (Supplementary Fig. 1a). We even could not detect any Tenericutes in other 101 three samples. Thus we propose that extreme low abundance might be a major reason 102 limiting the isolation of bacteria from this phylum. To obtain further insights into the 103 Tenericutes in deep-sea sediments, we performed metagenomics sequencing of the 104 sample ZC3 and analyzed all the annotated genes. The results showed that sequences 105 associated with phylum Tenericutes represented 0.250% of all annotated bacterial 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.18.388454; this version posted November 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 106 sequences (Supplementary Fig. 1b), which was consistent with the OTUs result of the 107 ZC3 sample (Supplementary Fig. 1a). Of note, the proportion of genes associated with 108 Candidatus Izimaplasma to those related to Tenericutes was up to 93.289% 109 (Supplementary Fig. 1c), indicating the dominant status of Candidatus Izimaplasma 110 within the deep-sea Tenericutes. 111 DNA degradation-driven