Johanna Schmidt

Complete ecological isolation and cryptic diversity in Polynucleobacter bacteria not resolved by 16S rRNA gene sequences

Transplantation experiments and genome comparisons were used to determine if lineages of planktonic Polynucleobacter almost indistinguishable by their 16S ribosomal RNA (rRNA) sequences differ distinctively in their ecophysiological and genomic traits. The results of three transplantation experiments differing in complexity of biotic interactions revealed complete ecological isolation between some of the lineages. This pattern fits well to the previously detected environmental distribution of lineages along chemical gradients, as well as to differences in gene content putatively providing adaptation to chemically distinct habitats. Patterns of distribution of iron transporter genes across 209 Polynucleobacter strains obtained from freshwater systems and representing a broad pH spectrum further emphasize differences in habitat-specific adaptations. Genome comparisons of six strains sharing ⩾99% 16S rRNA similarities suggested that each strain represents a distinct species. Comparison of sequence diversity among genomes with sequence diversity among 240 cultivated Polynucleobacter strains indicated a large cryptic species complex not resolvable by 16S rRNA sequences. The revealed ecological isolation and cryptic diversity in Polynucleobacter bacteria is crucial in the interpretation of diversity studies on freshwater bacterioplankton based on ribosomal sequences.

Rhodoluna lacicola gen. nov., sp. nov., a planktonic freshwater bacterium with stream-lined genome

A pure culture of an actinobacterium previously described as ‘Candidatus Rhodoluna lacicola’ strain MWH-Ta8 was established and deposited in two public culture collections. Strain MWH-Ta8T represents a free-living planktonic freshwater bacterium obtained from hypertrophic Meiliang Bay, Lake Taihu, PR China. The strain was characterized by phylogenetic and taxonomic investigations, as well as by determination of its complete genome sequence. Strain MWH-Ta8T is noticeable due to its unusually low values of cell size (0.05 µm3), genome size (1.43 Mbp), and DNA G+C content (51.5 mol%). Phylogenetic analyses based on 16S rRNA gene and RpoB sequences suggested that strain MWH-Ta8T is affiliated with the family Microbacteriaceae with Pontimonas salivibrio being its closest relative among the currently described species within this family. Strain MWH-Ta8T and the type strain of Pontimonas salivibrio shared a 16S rRNA gene sequence similarity of 94.3 %. The cell-wall peptidoglycan of strain MWH-Ta8T was of type B2β (B10), containing 2,4-diaminobutyric acid as the diamino acid. The predominant cellular fatty acids were anteiso-C15 : 0 (36.5 %), iso-C16 : 0 (16.5 %), iso-C15 : 0 (15.6 %) and iso-C14 : 0 (8.9 %), and the major (>10 %) menaquinones were MK-11 and MK-12. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and two unknown glycolipids. The combined phylogenetic, phenotypic and chemotaxonomic data clearly suggest that strain MWH-Ta8T represents a novel species of a new genus in the family Microbacteriaceae, for which the name Rhodoluna lacicola gen. nov., sp. nov. is proposed. The type strain of the type species is MWH-Ta8T ( = DSM 23834T = LMG 26932T).

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.

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 Polynucleobacter necessarius 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.

Microdiversification of a Pelagic Polynucleobacter Species Is Mainly Driven by Acquisition of Genomic Islands from a Partially Interspecific Gene Pool

ABSTRACT Microdiversification of a planktonic freshwater bacterium was studied by comparing 37 Polynucleobacter asymbioticus strains obtained from three geographically separated sites in the Austrian Alps. Genome comparison of nine strains revealed a core genome of 1.8 Mb, representing 81% of the average genome size. Seventy-five percent of the remaining flexible genome is clustered in genomic islands (GIs). Twenty-four genomic positions could be identified where GIs are potentially located. These positions are occupied strain specifically from a set of 28 GI variants, classified according to similarities in their gene content. One variant, present in 62% of the isolates, encodes a pathway for the degradation of aromatic compounds, and another, found in 78% of the strains, contains an operon for nitrate assimilation. Both variants were shown in ecophysiological tests to be functional, thus providing the potential for microniche partitioning. In addition, detected interspecific horizontal exchange of GIs indicates a large gene pool accessible to Polynucleobacter species. In contrast to core genes, GIs are spread more successfully across spatially separated freshwater habitats. The mobility and functional diversity of GIs allow for rapid evolution, which may be a key aspect for the ubiquitous occurrence of Polynucleobacter bacteria. IMPORTANCE Assessing the ecological relevance of bacterial diversity is a key challenge for current microbial ecology. The polyphasic approach which was applied in this study, including targeted isolation of strains, genome analysis, and ecophysiological tests, is crucial for the linkage of genetic and ecological knowledge. Particularly great importance is attached to the high number of closely related strains which were investigated, represented by genome-wide average nucleotide identities (ANI) larger than 97%. The extent of functional diversification found on this narrow phylogenetic scale is compelling. Moreover, the transfer of metabolically relevant genomic islands between more distant members of the Polynucleobacter community provides important insights toward a better understanding of the evolution of these globally abundant freshwater bacteria.

Polynucleobacter wuianus sp. nov., a free-living freshwater bacterium affiliated with the cryptic species complex PnecC

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.

Polynucleobacter sphagniphilus sp. nov. a planktonic freshwater bacterium isolated from an acidic and humic freshwater habitat

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 : 1ω7c, C16 : 0 and C18 : 1ω7c. 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.

Polynucleobacter aenigmaticus sp. nov. isolated from the permanently anoxic monimolimnion of a temperate meromictic lake

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 : 1ω7c, C18 : 1ω7c 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.

Silvanigrella aquatica gen. nov., sp. nov., isolated from a freshwater lake, description of Silvanigrellaceae fam. nov. and Silvanigrellales ord. nov., reclassification of the order Bdellovibrionales in the class Oligoflexia, reclassification of the families Bacteriovoracaceae and Halobacteriovoraceae in the new order Bacteriovoracales ord. nov., and reclassification of the family Pseudobacteriovoracaceae in the order Oligoflexales.

The unusual chemo-organoheterotrophic proteobacterial strain MWH-Nonnen-W8redT was isolated from a lake located in the Black Forest (Schwarzwald), Germany, by using the filtration-acclimatization method. Phylogenetic analyses based on the 16S rRNA gene sequence of the strain could not provide clear hints on classification of the strain in one of the current classes of the phylum Proteobacteria. Whole-genome sequencing resulted in a genome size of 3.5 Mbp and revealed a quite low DNA G+C content of 32.6 mol%. In-depth phylogenetic analyses based on alignments of 74 protein sequences of a phylogenetically broad range of taxa suggested assignment of the strain to a new order of the class Oligoflexia. These analyses also suggested that the order Bdellovibrionales should be transferred from the class Deltaproteobacteria to the class Oligoflexia, that this order should be split into two orders, and that the family Pseudobacteriovoracaceae should be transferred from the order Bdellovibrionales to the order Oligoflexales. We propose to establish for strain MWH-Nonnen-W8redT (=DSM 23856T=CCUG 58639T) the novel species and genus Silvanigrella aquatica gen. nov., sp. nov. to be placed in the new family Silvanigrellaceae fam. nov. of the new order Silvanigrellales ord. nov.

Reclassification of a Polynucleobacter cosmopolitanus strain isolated from tropical Lake Victoria as Polynucleobacter victoriensis sp. nov.

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).

Polynucleobacter meluiroseus sp. nov., a bacterium isolated from a lake located in the mountains of the Mediterranean island of Corsica

Strain AP-Melu-1000-B4 was isolated from a lake located in the mountains of the Mediterranean island of Corsica (France). Phenotypic, chemotaxonomic and genomic traits were investigated. Phylogenetic analyses based on 16S rRNA gene sequencing referred the strain to the cryptic species complex PnecC within the genus Polynucleobacter. The strain encoded genes for biosynthesis of proteorhodopsin and retinal. When pelleted by centrifugation the strain showed an intense rose colouring. Major fatty acids were C16 : 1ω7c, C16 : 0, C18 : 1ω7c and summed feature 2 (C16 : 1 isoI and C14 : 0-3OH). The sequence of the 16S rRNA gene contained an indel which was not present in any previously described Polynucleobacter species. Genome sequencing revealed a genome size of 1.89 Mbp and a G+C content of 46.6 mol%. In order to resolve the phylogenetic position of the new strain within subcluster PnecC, its phylogeny was reconstructed from sequences of 319 shared genes. To represent all currently described Polynucleobacter species by whole genome sequences, three type strains were additionally sequenced. Our phylogenetic analysis revealed that strain AP-Melu-100-B4 occupied a basal position compared with previously described PnecC strains. Pairwise determined whole genome average nucleotide identity (gANI) values suggested that strain AP-Melu-1000-B4 represents a new species, for which we propose the name Polynucleobacter meluiroseus sp. nov. with the type strain AP-Melu-1000-B4T (=DSM 103591T=CIP 111329T).