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Emended description of the genus Polynucleobacter and the species Polynucleobacter necessarius and proposal of two subspecies, P. necessarius subsp. necessarius subsp. nov. and P. necessarius subsp. asymbioticus subsp. nov.

¹ Institute for Limnology, Austrian Academy of Sciences, Mondseestrasse 9, 5310 Mondsee, Austria
² DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße 7B, D-38124 Braunschweig, Germany
³ State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, East Beijing Road 73, Nanjing 210008, PR China

Correspondence
Martin W. Hahn
martin.hahn{at}oeaw.ac.at

Heckmann and Schmidt described the genus Polynucleobacter for bacterial endosymbionts of freshwater ciliates affiliated with the genus Euplotes, and the species Polynucleobacter necessarius for obligate endosymbionts living in the cytoplasm of Euplotes aediculatus. Pure cultures of the type strain could not be established due to the obligate nature of the symbiotic relationship between the endosymbionts and their hosts. Therefore, Polynucleobacter necessarius is one of a few bacterial species with validly published names that lack a deposited pure culture. Meanwhile, it was demonstrated that the endosymbionts used for the description of the type of Polynucleobacter necessarius are closely related to obligately free-living strains. Similarity values of the 16S rRNA gene sequences obtained from the endosymbionts in the ciliate culture and free-living isolates in the range 99.1–99.4 % indicate that these organisms belong to the same species. Here, we have emended the description of Polynucleobacter necessarius by characterization of free-living strains maintained as pure cultures. The species Polynucleobacter necessarius was characterized as having low G+C contents of the DNA (44–46 mol%), small genome sizes (1.5–2.5 Mbp) and a lack of motility. Because of distinct differences in lifestyle and the genome size of Polynucleobacter necessarius strains, we propose that two novel subspecies should be established, Polynucleobacter necessarius subsp. necessarius subsp. nov. [with a type, which is a description based on endosymbionts in the culture ‘stock 15’ of the ciliate E. aediculatus (ATCC 30859)] and Polynucleobacter necessarius subsp. asymbioticus subsp. nov. [with the type strain QLW-P1DMWA-1^T (=DSM 18221^T=CIP 109841^T)], for the obligate endosymbionts of E. aediculatus and Euplotes harpa and obligately free-living strains, respectively.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains QLW-P1DMWA-1^T, MWH-JaK3, MWH-MoK4 and MWH-HuW1 are AJ879783, AJ550657, AJ550654 and AJ550666, respectively.

Images of colonies formed by a pure culture of a free-living Polynucleobacter strain and a graph showing the maximum optical density in NSY medium of the strains described, related free-living strains and the type strains of four other species affiliated with the family Burkholderiaceae, and a table giving the habitats from which the free-living strains investigated were isolated are available as supplementary material with the online version of this paper.

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