key: cord-0304772-nt6t2rff authors: Brooke, Charles; Connolly, Morgan P.; Garcia, Javier A.; Harmon-Smith, Miranda; Shapiro, Nicole; Hawley, Erik; Barton, Michael; Tringe, Susannah G.; Glavina del Rio, Tijana; Culley, David E.; Castenholz, Richard; Hess, Matthias title: Community structure of phototrophic co-cultures from extreme environments date: 2018-10-03 journal: bioRxiv DOI: 10.1101/427211 sha: 3be9a67a78e34a01b2b7b173a097630f1fd85c6f doc_id: 304772 cord_uid: nt6t2rff Cyanobacteria are found in most illuminated environments and are key players in global carbon and nitrogen cycling. Although significant efforts have been made to advance our understanding of this important phylum, still little is known about how members of the cyanobacteria affect and respond to changes in complex biological systems. This lack of knowledge is in part due to the reliance on our ability to maintain pure cultures when determining the metabolism and function of a microorganism. To fill this knowledge-gap, we selected 26 photosynthetic co-cultures from the Culture Collection of Microorganisms from Extreme Environments (CCMEE) for 16S rRNA gene sequencing. We assessed if samples readily available from the CCMEE could contribute valuable insights to advance applied and fundamental science in the areas of global and local carbon and nitrogen cycling, without growing individual members of these co-cultures axenically. Results from this work will aid in determining whether culture depositories in general hold the potential to advance fundamental and applied research. Since maintaining culture depositories is resource intensive, such an assessment will be of great value in guiding future funding decisions. Cyanobacteria are photosynthetic prokaryotes that are found in the majority of illuminated 44 habitats and are known to be some of the most morphologically diverse prokaryotes on 45 our planet 1 . The global cyanobacterial biomass is estimated to total ~3x10 14 g of carbon 46 productivity 3 . The efficient photosynthetic machinery of cyanobacteria has inspired 48 growing interest in the utilization of axenic cyanobacteria as well as cyanobacteria 49 containing co-cultures in microbial fuel cells 4,5 . In addition to having a global effect on the 50 carbon cycle, cyanobacteria-mediated nitrogen fixation has been estimated to supply 20-51 50% of the nitrogen input in some marine environments 6 . A detailed comprehension of 52 cyanobacteria and their contribution to global carbon and nitrogen cycling is therefore 53 necessary for a multi-scalar understanding of these globally important nutrient cycles and 54 ultimately for our ability to build accurate models to predict future climate patterns. 55 56 Besides their ecological relevance, cyanobacteria have potential applications in 57 biotechnology. The photosynthetic metabolism of cyanobacteria facilitates the assimilation 58 of carbon dioxide, a cheap and abundant substrate, to synthesize a variety of value-added 59 compounds with industrial relevance 7 . Numerous strains of cyanobacteria have been 60 investigated for their potential to produce bioactive compounds, biofertilizer, biofuels, and 61 bioplastics 8 ; and interactions of cyanobacterial strains with other bacteria have been 62 found to improve desirable cyanobacterial phenotypes 9 . Genes encoding enzymes 63 capable of catalyzing reactions that result in unique products, such as modified trichamide, 64 a cyclic peptide suggested to protect the bloom-forming Trichodesmium erythraeum 65 against predation 10 , and prochlorosins, a family of lanthipeptides with diverse functions 66 that are synthesized by various strains of Prochlorococcus and Synechococcus 11,12 , have 67 been identified from cyanobacterial genomes 13, 14 . It is very likely that de novo genome 68 assembly from metagenomic data will facilitate the discovery of novel enzymes from 69 cyanobacteria for which we currently lack the appropriate isolation and cultivation 70 techniques. Although metagenome-derived genomes hold great potential to enhance our 71 knowledge about genomic dark matter, ultimately, improved techniques to isolate and 72 enable axenic culturing of microorganisms that are currently characterized as 73 subsurface (1-5 mm depths) travertine deposits in YNP 20 In order to visualize the overall compositional differences between the co-cultures, an 199 uncorrected pairwise distance matrix was generated using the dist.seqs command in 200 MOTHUR and a tree was generated using Clearcut (version 1.0.9) 33 . A cladogram from 201 the resulting tree file was constructed and visualized using iTOL (https://itol.embl.de; 202 accessed on October 16 th , 2016; 34 ). Cluster designations were assigned at a branch 203 length of 0.05. Samples whose branches split at a distance >0.05 were considered as 204 part of the same cluster ( Figure 2 ~3.8% (±0.57%) of the raw reads from each sample due to insufficient quality. The 219 remaining reads were assigned to a total of 5,785 distinct Operational Taxonomic Units 220 (OTUs) based on 97% sequence identity (Table S2) . 221 To estimate the microbial diversity within each sample, rarefaction analyses were 223 performed (Supplemental Figure S1 ) and diversity indices were calculated ( Table 2 ). The 224 inverse Simpson index of the samples ranged between 1.52 and 9.24 with the lowest and 225 highest indices calculated for FECB3 and FECB32 respectively ( Table S2 ). Both OTU000015 and OTU000061 were classified as 243 Rhodobacteriaceae and recruited 9.2% and 8.2% of the reads generated for FECB3. 244 Whereas a taxonomic classification of OTU000015 was not possible at a resolution higher 245 than the family level, OTU000061 was classified as Paracoccus marcusii, a Gram negative 246 organism that displays a bright orange color due to the synthesis of carotenoids such as 247 astaxanthin 37 Community composition analysis revealed that each of the co-cultures contained at least 261 one OTU (mean (SD) = 2 (±1.23)) that recruited >0.1% of the co-culture specific reads 262 and that was classified as Cyanobacteria (Table 3 ). The only other phylum present in 263 each of the individual 26 co-cultures and represented by at least one OTU recruiting 264 >0.1% of the reads was the Proteobacteria phylum (Table 3 ). In contrast, only three 265 samples, namely FECB5, FECB30 and FECB68, contained OTUs that recruited >0.1% 266 of the sample specific reads and that could not be classified at the phylum level or at a 267 higher taxonomic resolution (Table 3 ). It is possible that the relatively high abundance of 268 non-classified phyla might contribute to the separation of these samples into distinct 269 clusters (i.e. cluster XII, IX, and IV) ( Figure 2 ). In addition to their omnipresence, 270 Cyanobacteria and Proteobacteria also recruited the majority of the reads in all but four 271 Table S2 ). Alicyclobacillus tolerans and Geobacillus vulcani have been 295 described previously as aerobic spore-forming thermophiles and have been isolated 296 from lead-zinc ores 38 and hot springs 39 in Russia, respectively. Members of the genus 297 Paenibacillus have been isolated from a wide variety of environments and some 298 Paenibacillus species have been found to promote crop growth directly via biological 299 nitrogen fixation, phosphate solubilization, production of the phytohormone indole-3-300 acetic acid and they have been identified as a potential source of novel antimicrobial 301 agents 40 . Although it is difficult to make a reliable prediction of the metabolic capacities 302 of the organism associated with OTU000082 solely based on 16S rRNA data, it is 303 certainly possible that this organism might possess the ability to promote or inhibit plant 304 and microbial growth respectively. 305 306 Photosynthetic co-cultures from Antarctica and YNP to study adaptation to increased 307 radiation, low temperatures and oligotrophic growth conditions 308 Microbial adaptation to extreme environments and the molecular framework that allows 309 microorganisms to survive and thrive in the presence of increased rates of radiation, low 310 temperatures and in the absence of nutrients has fascinated the scientific community for 311 decades and remains poorly understood. In an attempt to provide a better basis of the 312 taxonomic make-up of co-cultures that were collected from ecosystems that are 313 characterized by these extremes we included co-cultures from Antarctica Phylogenetic analysis of the heterotrophic population associated with FECB32, which 355 was isolated from travertine deposited by hot springs in YNP, found that sequences from 356 MLE-12 (OTU000109) recruited ~2% of the sample specific sequences (Supplemental 357 Table S2 ). This rendered MLE-12, previously assigned to the deep-branching candidate 358 phylum Melainabacteria 52 , as the eleventh most abundant organism in this 359 photosynthetic co-culture. It has been proposed previously that Melainabacteria, which is 360 commonly found in aquatic habitats, separated from the cyanobacteria before the latter 361 acquired photosynthetic capabilities 52 . Hence FECB32 might be a particularly valuable 362 co-culture to generate new insights into the evolution of and relationship between the 363 phylogenetically closely related Cyanobacteria and Melainabacteria. In addition, this 364 sample might provide the opportunity to enhance our understanding of the origin of 365 oxygenic photosynthesis and aerobic respiration in Cyanobacteria, an area that is 366 currently still poorly understood 53 . 367 368 Interestingly, OTU000109 was also detected in FECB36 and FECB38 (Supplemental 369 Table S2 ), although at significantly lower abundance (<0.001%). FECB36 and FECB38 370 were similar to FECB32 in that they were isolated from sites in YNP. Interestingly, 371 FECB32 and FECB38 cluster together (cluster IX) suggesting a similar overall microbial 372 community profiles, but separately from FECB36 (Figure 2 ). The only additional samples 373 that contained OTUs classified as Melainabacteria, recruiting >0.1% of the generated 374 reads, were FECB58 and FECB68 with ~0.9% and ~0.2% of their reads to this deeply 375 branched phylum, respectively (Supplemental Table S2 ). It seems noteworthy that 376 FECB58 and FECB68 were also isolated from hot springs and clustered closely together 377 based on their overall microbiome composition (Clusters V and IV respectively; Figure 378 2 contained only 16 OTUs that recruited more than 0.1% of the reads each (Table S2) Information about a Co-Culture Sample (MICCS) would be a significant step in 517 standardizing sample acquisition and maintenance, increasing the value of current and 518 future microbial samples collected from the environment. Developing MICCS and applying 519 them to co-cultures currently available from existing culture depositories is beyond the 520 scope of the work presented here, but we hope that the results presented here will 521 contribute to the initiation of this process and stimulate broad involvement and support 522 from the scientific community and various funding agencies. 523 524 Another noteworthy aspect of samples readily available through existing culture 525 collections, including the consortia discussed in this work, is their educational value. More 526 specifically, samples that can be acquired and maintained without the need of significant 527 resources and for which basic phylogenetic and functional information is available. These 528 co-cultures provide a unique opportunity for exciting undergraduate research, in 529 combining microbial diversity, microbial ecology and biotechnology. Techniques for basic 530 biochemical and physiological characterizations of these samples could be learned and 531 conducted by dedicated undergraduate students within a few weeks. A research program 532 based on these co-cultures would provide students with the unique opportunity to develop 533 laboratory skills and to learn firsthand about biogeochemical processes that shape our 534 environment and climate. Additional publicly available omics data, such as metagenomics 535 and metatranscriptomics generated from individual samples, would extend the scope of 536 these undergraduate research programs, in providing students the opportunity to learn 537 various omics analysis techniques using web-based tools or standalone scripts, 538 depending on the educational level and interest of each student. 539 540 In summary, culture collections that provide access to and standardized information 541 about microorganisms and microbial consortia provide opportunities for educational and 542 scientific progress. Therefore, it is of high importance that culture collections continue to 543 obtain the financial support necessary to provide this invaluable service to our society 544 Construction and characteristics of artificial consortia of 568 Scenedesmus obliquus-bacteria for S. obliquus growth and lipid production Structure of 571 trichamide, a cyclic peptide from the bloom-forming cyanobacterium 572 Trichodesmium erythraeum, predicted from the genome sequence Catalytic promiscuity in the biosynthesis of cyclic peptide secondary 575 metabolites in planktonic marine cyanobacteria Evolutionary radiation of lanthipeptides in marine cyanobacteria Unique marine 582 derived cyanobacterial biosynthetic genes for chemical diversity Cyanobacterial-based 585 approaches to improving photosynthesis in plants A Review of Living Collections with Special Emphasis on 588 Sustainability and Its Impact on Research Across Multiple Disciplines The United States Culture Collection Network (USCCN): 591 Enhancing Microbial Genomics Research through Living Microbe Culture 592 Evolution of thermotolerance in hot spring 595 cyanobacteria of the genus Synechococcus EVOLUTIONARY 598 RELATIONSHIPS OF CULTIVATED ANTARCTIC OSCILLATORIANS 599 (CYANOBACTERIA) CHARACTERIZATION OF PSYCHROPHILIC 602 OSCILLATORIANS (CYANOBACTERIA) FROM ANTARCTIC MELTWATER 603 PONDS Endolithic photosynthetic communities within 606 ancient and recent travertine deposits in Yellowstone National Park 609 Biogeographic and phylogenetic diversity of thermoacidophilic cyanidiales in The synthesis of the UV-screening pigment, 613 scytonemin, and photosynthetic performance in isolates from closely related 614 natural populations of cyanobacteria (Calothrix sp Effect of 617 environmental factors on the synthesis of scytonemin, a UV-screening pigment, 618 in a cyanobacterium (Chroococcidiopsis sp.) 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We would also thank Drs UC Davis for providing valuable comments and 772 suggestions on how to improve this manuscript Richard Castenholz who 774 passed away during the completion of this work after a long and satisfying journey in the 775 world of Cyanobacteria. He was, and will remain, a great inspiration to many of us Conflict of Interest: The authors declare no conflicts of interest Tringe wrote the manuscript. Richard Castenholz and Matthias Hess designed the 782 experiment. Erik Hawley and Matthias Hess performed experiment Tringe generated the data