Polynucleobacter necessarius, a model for genome reduction in both free-living and symbiotic bacteria Vittorio Boscaroa,1, Michele Fellettia,1,2, Claudia Vanninia, Matthew S. Ackermanb, Patrick S. G. Chainc, Stephanie Malfattid, Lisa M. Vergeze, Maria Shinf, Thomas G. Doakb, Michael Lynchb, and Giulio Petronia,3 aDepartment of Biology, Pisa University, 56126 Pisa, Italy; bDepartment of Biology, Indiana University, Bloomington, IN 47401; cLos Alamos National Laboratory, Los Alamos, NM 87545; dJoint Genome Institute, Walnut Creek, CA 94598; eLawrence Livermore National Laboratory, Livermore, CA 94550; and fEureka Genomics, Hercules, CA 94547 Edited by Nancy A. Moran, University of Texas at Austin, Austin, TX, and approved October 1, 2013 (received for review September 6, 2013) We present the complete genomic sequence of the essential belonging to free-living freshwater bacteria came as a surprise. symbiont Polynucleobacter necessarius (Betaproteobacteria), These free-living strains, which have been isolated and cultivated which is a valuable case study for several reasons. First, it is hosted (17), are ubiquitous and abundant in the plankton of lentic by a ciliated protist, Euplotes; bacterial symbionts of ciliates are environments (17, 18). They are smaller and do not show the still poorly known because of a lack of extensive molecular data. most prominent morphological feature of the symbiotic form: Second, the single species P. necessarius contains both symbiotic the presence of multiple nucleoids, each containing one copy of and free-living strains, allowing for a comparison between closely the genome (10, 11). It is clear that free-living and endosymbiotic related organisms with different ecologies. Third, free-living P. necessarius are not different life stages of the same organism P. necessarius strains are exceptional by themselves because of (15). Nevertheless, these strikingly different bacteria, occupying their small genome size, reduced metabolic flexibility, and high separate ecological niches, exhibit >99% 16S rRNA gene se- worldwide abundance in freshwater systems. We provide a com- quence identity, and phylogenetic analyses fail to separate them parative analysis of P. necessarius metabolism and explore the into two distinct groups (15). Rather, several lines of evidence peculiar features of a genome reduction that occurred on an point to multiple, recent origins of symbiotic strains from the already streamlined genome. We compare this unusual system free-living bacterial pool (14, 15). with current hypotheses for genome erosion in symbionts and Thus, the Euplotes–Polynucleobacter symbiosis provides a prom- free-living bacteria, propose modifications to the presently accepted ising system for the study of changes promoting or caused by the model, and discuss the potential consequences of translesion DNA shifttoanintracellularlifestyle. The remarkably small (2.16 Mbp) polymerase loss. genome of the free-living strain QLW-P1DMWA-1 has been se- quenced and studied, especially for features that would explain the symbiosis | nonsynonymous mutation rates | Burkholderiales | protozoa | success of this lineage in freshwater systems worldwide (19, 20). genome streamlining Phylogenies based on the 16S rRNA gene (13, 14) and multiple- gene analyses (19, 21, 22) consistently cluster Polynucleobacter Burkholderiaceae Betaproteobacteria ymbiosis, defined as a close relationship between organisms with bacteria of the family ( ), Ralstonia belonging to different species (1), is a ubiquitous, diverse, either in a basal position or as the sister group of S Cupriavidus and important mechanism in ecology and evolution (e.g., refs. 2– and . 4). In extreme cases, through the establishment of symbiotic relationships, quite unrelated lineages can functionally combine Significance their genomes and generate advantageous emergent features or initiate parasite/host arms races. Ciliates, common unicellular We have investigated multiple aspects of the Euplotes-Poly- protists of the phylum Ciliophora, are extraordinary receptacles nucleobacter system, which provides a unique opportunity for prokaryotic ecto- and endosymbionts (5, 6) that provide for the study of an obligate symbiont with a closely related varied examples of biodiversity and ecological roles (6). Never- free-living organism that itself possesses a peculiarly reduced theless, most of these symbionts are understudied, partially ow- genome and metabolism. We confirmed the robustness and ing to the scarcity of available molecular data and the absence of generality of patterns in the evolution of bacterial symbionts’ sequenced genomes. Yet, thanks to their various biologies and genome, adding at the same time new elements and hypotheses the ease of sampling and cultivating their protist hosts, they are concerning genome reduction in both symbiotic and free-living excellent potential models for symbioses between bacteria and bacteria. We argue that this system will provide an exceptionally heterotrophic eukaryotes. Until recently this field was dominated useful model for investigations on symbiosis, because of its by studies on endosymbionts of invertebrates, especially insects peculiarities and the commonness and ease of handling of the (e.g., ref. 7), although unicellular systems like amoebas (e.g., refs. ciliate hosts. Genome sequences for independently derived 8 and 9) have been shown to be suitable models. Polynucleobacter symbionts will be particularly telling. Polynucleobacter necessarius was first described as a cytoplas- mic endosymbiont of the ciliate Euplotes aediculatus (10, 11). Author contributions: C.V., M.L., and G.P. designed research; C.V., P.S.G.C., S.M., L.M.V., Further surveys detected its presence in a monophyletic group and M.S. performed research; V.B., M.F., M.S.A., P.S.G.C., S.M., L.M.V., M.S., and T.G.D. of fresh and brackish water Euplotes species (12, 13). All of the analyzed data; V.B. and M.F. wrote the paper; and G.P. coordinated research. investigated strains of these species die soon after being cured The authors declare no conflict of interest. of the endosymbiont (10, 12, 13). In the few cases in which This article is a PNAS Direct Submission. P. necessarius is not present, a different and rarer bacterium Data deposition: The sequence reported in this paper has been deposited in the GenBank apparently supplies the same function (12, 14). No attempt to grow database (accession no. NC_010531.1). symbiotic P. necessarius outside their hosts has yet been suc- 1V.B. and M.F. contributed equally to this work. cessful (15), strongly suggesting that the relationship is obligate 2Present address: Department of Chemistry, University of Konstanz, 78457 Konstanz, for both partners, in contrast to most other known prokaryote/ Germany. ciliate symbioses (6). 3To whom correspondence should be addressed. E-mail: [email protected]. Thus, the findings of many environmental 16S rRNA gene This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sequences similar to that of the symbiotic P. necessarius (16) but 1073/pnas.1316687110/-/DCSupplemental. 18590–18595 | PNAS | November 12, 2013 | vol. 110 | no. 46 www.pnas.org/cgi/doi/10.1073/pnas.1316687110 Downloaded by guest on September 25, 2021 Here we provide the complete genomic sequence of a symbi- living relative) have unknown functions (SI Results and Discus- otic P. necessarius harbored in the cytoplasm of E. aediculatus sion, Genome Composition Analysis). and present a comparative analysis of the two sequenced Poly- nucleobacter genomes, addressing the possible biological basis of the Metabolism. Central metabolism and carbon sources. Both the symbi- Euplotes–Polynucleobacter symbiosis. We also provide insights into ont and the free-living P. necessarius lack a glycolytic pathway. the evolution of the unique two-step genome reduction in this They do not possess the central regulatory enzyme of the Embden- bacterialspecies:thefirst step involving streamlining in a free-living Meyerhof pathway (6-phosphofructokinase) nor enzymes spe- ancestor and the second a more recent period of genome erosion cific to the Entner-Doudoroff variant, which is used by most confined to the symbiotic lineage. Burkholderiaceae bacteria. They also lack any enzyme that could phosphorylate glucose to glucose-6-phosphate or be involved in Results and Discussion the assimilation of other monosaccharides. The inability to ex- General Features of the Genome. The circular chromosome (Fig. ploit sugars as carbon or energy sources reflects a general poverty S1) of the symbiotic P. necessarius strain is 1.56 Mbp long and in catabolic pathways. contains approximately 1,279 protein-coding genes (SI Results Genomic analysis (Fig. 2 and SI Results and Discussion, Details and Discussion, Genome Composition Analysis and Table S1). on Metabolic Analysis; see also ref. 19) suggests that the principal The reduction in genome size that has occurred since the es- carbon sources for the free-living strain are pyruvate, acetate, tablishment of symbiosis can be estimated by comparing this carboxylic acids, and probably compounds convertible to them. genome with that of the P. necessarius free-living strain QLW- These can be metabolized to acetyl-CoA, which is the key in- P1DMWA-1 (see also Fig. S2), an acceptable procedure given termediate of both energy production and anabolism, thanks to that the symbiont’s gene set is largely a subset of that of the free- a complete glyoxylate cycle, tricarboxylic acids cycle (TCA), and