Reclassification of four Polynucleobacter necessarius strains as representatives of Polynucleobacter asymbioticus comb. nov., Polynucleobacter duraquae sp. nov., Polynucleobacter yangtzensis sp. nov. and Polynucleobacter sinensis sp. nov., and emended description of Polynucleobacter necessarius.
ABSTRACT: Genome comparisons based on average nucleotide identity (ANI) values of four strains currently classified as Polynucleobacter necessarius subsp. asymbioticus resulted in ANI values of 75.7-78.4?%, suggesting that each of those strains represents a separate species. The species P. necessarius was proposed by Heckmann and Schmidt in 1987 to accommodate obligate endosymbionts of ciliates affiliated with the genus Euplotes. The required revision of this species is, however, hampered by the fact, that this species is based only on a description and lacks a type strain available as pure culture. Furthermore, the ciliate culture Euplotes aediculatus ATCC 30859, on which the description of the species was based, is no longer available. We found another Euplotes aediculatus culture (Ammermann) sharing the same origin with ATCC 30859 and proved the identity of the endosymbionts contained in the two cultures. A multilocus sequence comparison approach was used to estimate if the four strains currently classified as Polynucleobacternecessarius subsp. asymbioticus share ANI values with the endosymbiont in the Ammermann culture above or below the threshold for species demarcation. A significant correlation (R2 0.98, P<0.0001) between multilocus sequence similarity and ANI values of genome-sequenced strains enabled the prediction that it is highly unlikely that these four strains belong to the species P. necessarius. We propose reclassification of strains QLW-P1DMWA-1T (=DSM 18221T=CIP 109841T), MWH-MoK4T (=DSM 21495T=CIP 110977T), MWH-JaK3T (=DSM 21493T=CIP 110976T) and MWH-HuW1T (=DSM 21492T=CIP 110978T) as Polynucleobacter asymbioticus comb. nov., Polynucleobacter duraquae sp. nov., Polynucleobacter yangtzensis sp. nov. and Polynucleobacter sinensis sp. nov., respectively.
Project description: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-1T (=DSM 18221T=CIP 109841T)], for the obligate endosymbionts of E. aediculatus and Euplotes harpa and obligately free-living strains, respectively.
Project description:Polynucleobacter necessarius subsp. asymbioticus strain QLW-P1DMWA-1(T) is a planktonic freshwater bacterium affiliated with the family Burkholderiaceae (class Betaproteobacteria). This strain is of interest because it represents a subspecies with cosmopolitan and ubiquitous distribution in standing freshwater systems. The 16S-23S ITS genotype represented by the sequenced strain comprised on average more than 10% of bacterioplankton in its home habitat. While all strains of the subspecies P. necessarius asymbioticus are free-living freshwater bacteria, strains belonging to the only other subspecies, P. necessarius subsp. necessarius are obligate endosymbionts of the ciliate Euplotes aediculatus. The two subspecies of P. necessarius are the instances of two closely related subspecies that differ in their lifestyle (free-living vs. obligate endosymbiont), and they are the only members of the genus Polynucleobacter with completely sequenced genomes. Here we describe the features of P. necessarius subsp. asymbioticus, together with the complete genome sequence and annotation. The 2,159,490 bp long chromosome with a total of 2,088 protein-coding and 48 RNA genes is the first completed genome sequence of the genus Polynucleobacter to be published and was sequenced as part of the DOE Joint Genome Institute Community Sequencing Program 2006.
Project description:Five heterotrophic, aerobic, catalase- and oxidase-positive, non-motile strains were characterized from freshwater habitats located in Austria, France, Uganda, P. R. China and New Zealand. The strains shared 16S rRNA gene similarities of >/=99.3 %. The novel strains grew on NSY medium over a temperature range of 10-35 degrees C (two strains also grew at 5 degrees C and one strain grew at 38 degrees C) and a NaCl tolerance range of 0.0-0.3 % (four strains grew up to 0.5 % NaCl). The predominant fatty acids were C(16 : 0), C(18 : 1)omega7c, C(12 : 0) 3-OH, and summed feature 3 (including C(16 : 1)omega7c). The DNA G+C content of strain MWH-MoIso2(T) was 44.9 mol%. Phylogenetic analysis of 16S rRNA gene sequences demonstrated that the five new strains formed a monophyletic cluster closely related to Polynucleobacter necessarius (96-97 % sequence similarity). This cluster also harboured other isolates as well as environmental sequences which have been obtained from several habitats. Investigations with taxon-specific FISH probes demonstrated that the novel bacteria dwell as free-living, planktonic cells in freshwater systems. Based on the revealed phylogeny and pronounced chemotaxonomic differences to P. necessarius (presence of >7 % C(12 : 0) 3-OH and absence of C(12 : 0) and C(12 : 0) 2-OH), the new strains are suggested to represent a novel species, for which the name Polynucleobacter cosmopolitanus sp. nov. is proposed. The type strain is MWH-MoIso2(T) (=DSM 21490(T)=CIP 109840(T)=LMG 25212(T)). The novel species belongs to the minority of described species of free-living bacteria for which both in situ data from their natural environments and culture-based knowledge are available.
Project description:Strains MWH-EgelM1-30-B4T and MWH-Feld-100T were isolated from the water columns of two freshwater systems. Both strains represent delicate bacteria not easy to work with in laboratory experiments. Phylogenetic analyses of the 16S rRNA genes suggested that both strains were affiliated with the genus Polynucleobacter. Both strains share 16S rRNA gene sequence similarities of >99?% with eight free-living Polynucleobacter type strains, all affiliated with the cryptic species complex PnecC. The full-length 16S rRNA gene sequences of the two strains differ only in two and three positions, respectively, from the sequence of the closest related Polynucleobacter type strain. Genome sequencing of both strains revealed relatively small genome sizes of 2.0 Mbp and G+C?contents of 45?mol%. Phylogenetic analyses based on nucleotide sequences of 319 shared protein-encoding genes consistently placed the two strains in taxon PnecC but did not suggest an affiliation with one of the previously described species. Pairwise analyses of whole genome average nucleotide identities (gANI) with representatives of all previously described Polynucleobacter species resulted in both cases throughout in values <80?%. Pairwise comparison of the genomes of the two new strains resulted in gANI values of 83.3?%. All gANI analyses clearly suggested that strains MWH-EgelM1-30-B4T and MWH-Feld-100T represent two novel Polynucleobacter species. We propose for these novel species the names Polynucleobacter hirudinilacicola sp. nov. and Polynucleobacter campilacus sp. nov. and strains MWH-EgelM1-30-B4T (=DSM 23911T=LMG 30144T) and MWH-Feld-100T (=DSM 24007T=LMG 29705T) as the type strains, respectively.
Project description:The genus Polynucleobacter (family Burkholderiaceae) is phylogenetically subdivided into at least four subclusters. One of those, subcluster PnecC, was recognized as a cryptic species complex. Here we test by comparative genome analyses whether subcluster PnecD, currently solely represented by the species Polynucleobactercosmopolitanus, also represents such a cryptic species complex. The genome sequences of the two P. cosmopolitanus strains, MWH-MoIso2T and MWH-VicM1, were determined. The latter strain was also characterized in the previous description of P. cosmopolitanus. These two strains originate from a temperate lake located in Austria and from the large tropical Lake Victoria located in East Africa, respectively. Strains MWH-MoIso2T and MWH-VicM1 possess quite small genomes of 1.78 and 1.63 Mbp, respectively, and share similar G+C?values of 44.1 and 43.1?mol%, respectively. Both strains encode only a single copy of the ribosomal operon, and their 16S rRNA genes differ only in four positions, equalling a sequence similarity of 99.74?%. Both genomes possess characteristics indicating evolutionary genome streamlining, such as high coding densities of 93.9 and 94.6?% of bases, respectively. Average nucleotide identity (ANI) comparisons of the genomes of the two strains resulted in a value of 78.4?%, suggesting that each of the strains represents a separate species. Our investigation suggests that PnecD represents an additional cryptic species complex within the genus Polynucleobacter that was not resolved by 16S rRNA gene sequence analyses. We propose reclassification of strain MWH-VicM1 as Polynucleobacter victoriensis sp. nov., with type strain MWH-VicM1T(=DSM 21486T=JCM 32005T).
Project description:The bacterial strain MWH-K35W1T was isolated from a permanently anoxic water layer of a meromictic lake located in the Austrian Salzkammergut area. The basically chemo-organoheterotrophic strain was isolated and maintained under aerobic conditions. Phylogenetic analyses of the 16S rRNA gene and the glutamine synthetase gene (glnA) of the strain suggested an affiliation to the genus Polynucleobacter and the cryptic species complex PnecC. Strain MWH-K35W1T shares with the type strains of the six free-living species of the genus Polynucleobacter affiliated with this species complex 16S rRNA gene sequence similarities of 99.6-99.9?%, while the type material of the obligate endosymbiont Polynucleobacternecessarius, which is also affiliated with this species complex, shares a gene sequence similarity of 99.1?%. Genome sequencing resulted in a genome size of 2.14 Mbp and a DNA G+C?content of 45.98?mol%. Major fatty acids were C16?:?1?7c, C18?:?1?7c and C16?:?0. This strain is the first strain of the genus Polynucleobacter found to encode a proteorhodopsin-like protein but, in contrast to some other strains affiliated to this genus, it does not encode a putative anoxygenic photosynthesis system. Multilocus sequence analysis based on partial sequences of eight housekeeping genes, as well as average nucleotide identity (ANI) analyses, did not suggest that strain MWH-K35W1T belongs to a previously described species. We propose the name Polynucleobacter aenigmaticus for a novel species with strain MWH-K35W1T (=DSM 24006T=LMG 29706T) as the type strain.
Project description:The heterotrophic, aerobic, facultatively anaerobic, catalase- and oxidase-positive, non-motile strain MWH-PoolGreenA3(T), isolated from a rock pool filled with freshwater, was characterized. The strain grew on NSY medium over a NaCl range of 0.0-0.3?% (w/v). Whole-cell fatty acids were dominated by C(16?:?1)?7c (feature 3), C(18?:?1)?7c and straight-chain C(16?:?0); furthermore, the components C(12?:?0) and C(14?:?0) 2-OH were present. The DNA G+C content was 48.3 mol%. Phylogenetic analysis as well as strong similarities in phenotypic and chemotaxonomic traits indicated the affiliation with the genus Polynucleobacter. 16S rRNA gene similarity values with the three described species of the genus Polynucleobacter ranged from 96.7 to 97.8?%. DNA-DNA hybridization experiments did not reveal that the strain belongs to a previously described species of the genus Polynucleobacter. The strain can be discriminated from previously established species of the genus Polynucleobacter by chemotaxonomic and phenotypic traits. The bacterium possesses a free-living lifestyle and represents a group of planktonic freshwater bacteria occurring with high cell numbers in many freshwater lakes. Based on the phylogeny revealed and the chemotaxonomic and phenotypic differences from previously described species of the genus Polynucleobacter, we propose to establish the novel species Polynucleobacter acidiphobus sp. nov. with the type strain MWH-PoolGreenA3(T) (?=?DSM 21994(T) ?=?CIP 110079(T)).
Project description:Strain MWH-Weng1-1T, isolated from an acidic freshwater habitat located in the Wenger Moor, Austria, was characterized by investigating its phenotypic, chemotaxonomic and genomic traits. Phylogenetic analyses based on 16S rRNA gene sequencing placed the strain in the cryptic species complex PnecC within the genus Polynucleobacter. The strain had a genome of 2.04 Mbp with a G+C content of 45.6?mol%. The major fatty acids of the strain were C16?:?1?7c, C16?:?0 and C18?:?1?7c. In order to resolve the systematic position of the strain within the species complex PnecC, concatenated partial sequences of eight housekeeping genes were used for phylogenetic analyses. The obtained trees did not place strain MWH-Weng1-1T close to any of the six previously described species within this cryptic species complex. Pairwise whole genome average nucleotide identity comparisons with genome sequences of strains representing the six previously described species of the subcluster resulted throughout in values <78?%, which clearly suggested that strain MWH-Weng1-1T (DSM 24018T=CIP 111099T) represents a novel species. We propose the name Polynucleobacter sphagniphilus sp. nov. and strain MWH-Weng1-1T as the type strain for this new species.
Project description:The heterotrophic, aerobic, facultatively anaerobic under denitrifying conditions, catalase- and oxidase-positive, non-motile strain MT-CBb6A5(T), which was isolated from an acidic lake located in Wisconsin (USA), was characterized. The strain grew on NSY medium over a temperature range of 15-30 °C and a NaCl range of 0.0-0.3?% (w/v). The predominant fatty acids were C(16?:?0), C(18?:?1)?7c, 11-methyl C(18?:?1)?7c, feature 3 (including C(16?:?1)?7c), and feature 2 (including C(14?:?0) 3-OH). The DNA G+C content of the strain was 40.3 mol%. Phylogenetic analysis as well as strong similarities in phenotypic and chemotaxonomic traits indicated the affiliation with the genus Polynucleobacter. 16S rRNA gene sequence similarity values with the two described species of the genus Polynucleobacter ranged from 95.6 to 96.0?%. The strain differs from the two described species of the genus Polynucleobacter in the ability to assimilate oxalic and glycolic acids, and in the presence of the fatty acids C(15?:?1)?8c and C(16?:?0) 3-OH as well as in quantitative differences in fatty acid composition. It has to be assumed that the strain shares with other free-living bacteria of the genus Polynucleobacter a planktonic lifestyle in the water column of freshwater habitats. Based on the phylogeny revealed and the chemotaxonomic and phenotypic differences from Polynucleobacter necessarius and Polynucleobacter cosmopolitanus, we propose to establish the novel species Polynucleobacter rarus sp. nov. with the type strain MT-CBb6A5(T) (?=?DSM 21648(T) ?=?CIP 109928(T)).
Project description:Strain QLW-P1FAT50C-4T, isolated from a shallow, acidic freshwater pond located in the Austrian Alps at an altitude of 1300 m, was characterized by investigation of phenotypic, chemotaxonomic and genomic traits. As shown previously, phylogenetic analyses based on 16S rRNA gene sequences placed the strain in the cryptic species complex PnecC within the genus Polynucleobacter. The major fatty acids of the strain were C16 : 1ω7c and C18 : 1ω7c. The strain has a genome of 2.23 Mbp with a DNA G+C content of 44.9 mol%. The strain encodes a seemingly complete gene cluster for anoxygenic photosynthesis but lacks typical genes for CO2 assimilation. In order to resolve the phylogenetic position of the strain within the species complex PnecC, concatenated partial sequences of eight housekeeping genes were analysed. The phylogenetic reconstruction obtained did not place strain QLW-P1FAT50C-4T close to any of the five previously described species within subcluster PnecC. Pairwise average nucleotide identity (ANI) comparisons of whole-genome sequences suggested that strain QLW-P1FAT50C-4T (=DSM 24008T=CIP 111100T) represents a novel species, for which we propose the name Polynucleobacter wuianus sp. nov.