Jitka Jezberová

The Passive Yet Successful Way of Planktonic Life: Genomic and Experimental Analysis of the Ecology of a Free-Living Polynucleobacter Population

Background The bacterial taxon Polynucleobacter necessarius subspecies asymbioticus represents a group of planktonic freshwater bacteria with cosmopolitan and ubiquitous distribution in standing freshwater habitats. These bacteria comprise <1% to 70% (on average about 20%) of total bacterioplankton cells in various freshwater habitats. The ubiquity of this taxon was recently explained by intra-taxon ecological diversification, i.e. specialization of lineages to specific environmental conditions; however, details on specific adaptations are not known. Here we investigated by means of genomic and experimental analyses the ecological adaptation of a persistent population dwelling in a small acidic pond. Findings The investigated population (F10 lineage) contributed on average 11% to total bacterioplankton in the pond during the vegetation periods (ice-free period, usually May to November). Only a low degree of genetic diversification of the population could be revealed. These bacteria are characterized by a small genome size (2.1 Mb), a relatively small number of genes involved in transduction of environmental signals, and the lack of motility and quorum sensing. Experiments indicated that these bacteria live as chemoorganotrophs by mainly utilizing low-molecular-weight substrates derived from photooxidation of humic substances. Conclusions Evolutionary genome streamlining resulted in a highly passive lifestyle so far only known among free-living bacteria from pelagic marine taxa dwelling in environmentally stable nutrient-poor off-shore systems. Surprisingly, such a lifestyle is also successful in a highly dynamic and nutrient-richer environment such as the water column of the investigated pond, which was undergoing complete mixis and pronounced stratification in diurnal cycles. Obviously, metabolic and ecological versatility is not a prerequisite for long-lasting establishment of abundant bacterial populations under highly dynamic environmental conditions. Caution should be exercised when generalizing the obtained insights into the ecology and adaptation of the investigated lineage to other Polynucleobacter lineages.

Ubiquity of Polynucleobacter necessarius subspecies asymbioticus results from ecological diversification

The subspecies Polynucleobacter necessarius asymbioticus (> 99% 16S rRNA similarity) has a cosmopolitan distribution and a ubiquitous occurrence in lentic freshwater habitats. We tested if the observed ubiquity of these free-living planktonic freshwater bacteria results from a euryoecious (generalist) adaptation of P. n. asymbioticus strains, or from ecological diversification within the subspecies. We developed a reverse line blot hybridization assay enabling the cultivation-independent detection of 13 groups within the subspecies in environmental samples. A set of 121 lentic freshwater habitats, spanning a broad variety of habitat types (e.g. pH levels ranging from 3.8 to 8.5) was investigated for the presence of these 13 P. n. asymbioticus groups. Statistical analyses of the reverse line blot hybridization detections revealed pronounced differences in habitat preferences of several of the groups. Their preferences differed regarding pH, conductivity, dissolved organic carbon and oxygen concentration of habitats. For some groups, differences in environmental preferences resulted even in complete niche separation between them. The revealed differences in habitat preferences suggest that the previously reported ubiquity of P. n. asymbioticus results from ecological diversification within the taxon and not from generalist adaptation of strains.

POLYPHASIC CHARACTERIZATION OF THREE STRAINS OF ANABAENA RENIFORMIS AND APHANIZOMENON APHANIZOMENOIDES (CYANOBACTERIA) AND THEIR RECLASSIFICATION TO SPHAEROSPERMUM GEN. NOV. (INCL. ANABAENA KISSELEVIANA)(1).

Occurrences of rare cyanobacteria Anabaena reniformis Lemmerm. and Aphanizomenon aphanizomenoides (Forti) Horecká et Komárek were recently detected at several localities in the Czech Republic. Two monoclonal strains of An. reniformis and one strain of Aph. aphanizomenoides were isolated from distant localities and different sampling years. They were characterized by a combination of morphological, genetic, and biochemical approaches. For the first time, partial 16S rRNA gene sequences were obtained for these morphospecies. Based on this gene, all of these strains clustered separately from other planktonic Anabaena and Aphanizomenon strains. They appeared in a cluster with Cylindrospermopsis Seenaya et Subba Raju and Raphidiopsis F. E. Fritsch et M. F. Rich, clustered closely together with two An. kisseleviana Elenkin strains available from GenBank. A new generic entity was defined (Sphaerospermum gen. nov., with the type species S. reniforme, based on the traditional species An. reniformis). These results contribute significantly to the knowledge base about genetic heterogeneity among planktonic Anabaena–like and Aphanizomenon–like morphospecies. Accordingly, the subgenus Dolichospermum, previously proposed for the group of planktonic Anabaena, should be revaluated. Secondary metabolite profiles of the An. reniformis and Aph. aphanizomenoides strains differed considerably from 17 other planktonic Anabaena strains of eight morphospecies isolated from Czech water bodies. Production of puwainaphycin A was found in both of the An. reniformis strains. Despite the relatively short phylogenetic distance from Cylidrospermopsis, the production of cylindrospermopsin was not detected in any of our strains.

Ubiquity of Polynucleobacter necessarius ssp asymbioticus in lentic freshwater habitats of a heterogenous 2000 km2 area

We present a survey on the distribution and habitat range of Polynucleobacter necessarius ssp. asymbioticus (PnecC), a numerically and functionally important taxon in the plankton of freshwater systems. We systematically sampled stagnant freshwater habitats in a heterogeneous 2000 km(2) area, together with ecologically different habitats outside this area. In total, 137 lakes, ponds and puddles were investigated, which represent an enormous diversity of habitats differing, e.g. in depth (< 10 cm-171 m) and pH (3.9-8.5). PnecC bacteria were detected by cultivation-independent methods in all investigated habitats, and their presence was confirmed by cultivation of strains from selected habitats representing the whole studied ecological range. The determined relative abundance of the subspecies ranged from values close to the detection limit of FISH (0.2%) to 67% (average 14.5%), and the highest observed absolute abundance was 5.3 x 10(6) cells ml(-1). Statistical analyses revealed that the abundance of PnecC bacteria was partially controlled by factors linked to concentrations of humic substances, which support the hypothesis that these bacteria utilize photodegradation products of humic substances. Based on the revealed statistical relationships, an average relative abundance of this subspecies of 20% in global freshwater habitats was extrapolated. Our study provides important implications for the current debate on ubiquity and biogeography in microorganisms.

Broad Habitat Range of the Phylogenetically Narrow R-BT065 Cluster, Representing a Core Group of the Betaproteobacterial Genus Limnohabitans

ABSTRACT The distribution of the phylogenetically narrow R-BT065 cluster (Betaproteobacteria) in 102 freshwater lakes, reservoirs, and various ponds located in central Europe (a total of 122 samples) was examined by using a cluster-specific fluorescence in situ hybridization probe. These habitats differ markedly in pH, conductivity, trophic status, surface area, altitude, bedrock type, and other limnological characteristics. Despite the broad ecological diversity of the habitats investigated, the cluster was detected in 96.7% of the systems, and its occurrence was not restricted to a certain habitat type. However, the relative proportions of the cluster in the total bacterioplankton were significantly lower in humic and acidified lakes than in pH-neutral or alkaline habitats. On average, the cluster accounted for 9.4% of the total bacterioplankton (range, 0 to 29%). The relative abundance and absolute abundance of these bacteria were significantly and positively related to higher pH, conductivity, and the proportion of low-molecular-weight compounds in dissolved organic carbon (DOC) and negatively related to the total DOC and dissolved aromatic carbon contents. Together, these parameters explained 55.3% of the variability in the occurrence of the cluster. Surprisingly, no clear relationship of the R-BT065 bacteria to factors indicating the trophic status of habitats (i.e., different forms of phosphorus and chlorophyll a content) was found. Based on our results and previously published data, we concluded that the R-BT065 cluster represents a ubiquitous, highly active segment of bacterioplankton in nonacidic lakes and ponds and that alga-derived substrates likely form the main pool of substrates responsible for its high growth potential and broad distribution in freshwater habitats.

Limnohabitans curvus gen. nov., sp. nov., a planktonic bacterium isolated from a freshwater lake

A chemo-organotrophic, aerobic, facultatively anaerobic, non-motile strain, MWH-C5(T), isolated from the water column of the oligomesotrophic Lake Mondsee (Austria), was characterized phenotypically, phylogenetically and chemotaxonomically. The predominant fatty acids of the strain were C(16 : 1)omega7c/omega6c, C(16 : 0), C(12 : 1) and C(8 : 0)-3OH, the major quinone was ubiquinone Q-8 and the G+C content of the DNA of the strain was 55.5 mol%. 16S rRNA gene similarity to the closest related type strains was 96.6 % (Curvibacter delicatus LMG 4328(T)) and 95.7 % (Rhodoferax fermentans FR3(T)). Phylogenetic analysis of 16S rRNA gene sequences revealed the affiliation of the strain with the family Comamonadaceae (Betaproteobacteria); however, the phylogenetic position of the strain did not support an affiliation to any previously described genus within this family. A family-wide comparison of traits revealed that the strain possesses a unique combination of DNA G+C content, major fatty acids and major 3-hydroxy fatty acid. Furthermore, the strain differs in several traits from the closest related genera. Based on the phylogeny of the strain and differences from closely related genera, we propose to establish the new genus and species Limnohabitans curvus gen. nov., sp. nov. to accommodate this strain. The type strain of Limnohabitans curvus is MWH-C5(T) (=DSM 21645(T) =CCUG 56720(T)). The type strain is closely related to a large number of uncultured bacteria detected by cultivation-independent methods in various freshwater systems.

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.

Polyphasic characterization of eight planktonic Anabaena strains (Cyanobacteria) with reference to the variability of 61 Anabaena populations observed in the field

The plasticity of morphological features used for single morphospecies identification was studied under varied experimental conditions (temperature, light, nitrogen, phosphorus) in eight planktonic Anabaena strains. The strains represented all of the morphospecies with coiled trichomes commonly occurring in Central Europe (two strains of A. mendotae & A. sigmoidea complex, two A. lemmermannii strains, two A. flos-aquae strains, and two strains of A. circinalis & A. crassa complex). Significant effects of the growth conditions on vegetative cell dimensions were observed in seven strains, and P concentration was the main influencing factor in most cases (six strains). Significant effect of an environmental factor (P) on akinete morphology was found in only one strain. Experimentally assessed temperature and light growth optima were specific for each strain and were not consistent with the taxonomic affiliation of the strains. Morphologies of the Anabaena strains studied were compared with the field morphologies of 61 Anabaena populations of eight morphospecies observed in the Czech Republic. The range of morphological variability of single strains under the experimental conditions spanned the total variability of the populations of relevant morphospecies observed in the field. Delimitations and proper descriptions of the morphospecies are discussed in the light of partial 16S rRNA gene sequences of the studied strains.

Patterns of Limnohabitans Microdiversity across a Large Set of Freshwater Habitats as Revealed by Reverse Line Blot Hybridization

Among abundant freshwater Betaproteobacteria, only few groups are considered to be of central ecological importance. One of them is the well-studied genus Limnohabitans and mainly its R-BT subcluster, investigated previously mainly by fluorescence in situ hybridization methods. We designed, based on sequences from a large Limnohabitans culture collection, 18 RLBH (Reverse Line Blot Hybridization) probes specific for different groups within the genus Limnohabitans by targeting diagnostic sequences on their 16 S–23 S rRNA ITS regions. The developed probes covered in sum 92% of the available isolates. This set of probes was applied to environmental DNA originating from 161 different European standing freshwater habitats to reveal the microdiversity (intra-genus) patterns of the Limnohabitans genus along a pH gradient. Investigated habitats differed in various physicochemical parameters, and represented a very broad range of standing freshwater habitats. The Limnohabitans microdiversity, assessed as number of RLBH-defined groups detected, increased significantly along the gradient of rising pH of habitats. 14 out of 18 probes returned detection signals that allowed predictions on the distribution of distinct Limnohabitans groups. Most probe-defined Limnohabitans groups showed preferences for alkaline habitats, one for acidic, and some seemed to lack preferences. Complete niche-separation was indicated for some of the probe-targeted groups. Moreover, bimodal distributions observed for some groups of Limnohabitans, suggested further niche separation between genotypes within the same probe-defined group. Statistical analyses suggested that different environmental parameters such as pH, conductivity, oxygen and altitude influenced the distribution of distinct groups. The results of our study do not support the hypothesis that the wide ecological distribution of Limnohabitans bacteria in standing freshwater habitats results from generalist adaptations of these bacteria. Instead, our observations suggest that the genus Limnohabitans, as well as its R-BT subgroup, represent ecologically heterogeneous taxa, which underwent pronounced ecological diversification.

POLYPHASIC CHARACTERIZATION OF DOLICHOSPERMUM SPP. AND SPHAEROSPERMOPSIS SPP. (NOSTOCALES, CYANOBACTERIA): MORPHOLOGY, 16S rRNA GENE SEQUENCES AND FATTY ACID AND SECONDARY METABOLITE PROFILES(1).

The genera Dolichospermum (Ralfs ex Bornet et Flahault) Wacklin, L. Hoffm. et Komárek and Sphaerospermopsis Zapomělová, Jezberová, Hrouzek, Hisem, K. Řeháková et Komárk.‐Legn. represent a highly diversified group of planktonic cyanobacteria that have been recently separated from the traditional genus Anabaena Bory ex Bornet et Flahault. In this study, morphological diversity, phylogeny of the 16S rRNA gene, production of fatty acids, and secondary metabolite profiles were evaluated in 33 strains of 14 morphospecies isolated from the Czech Republic. Clustering of the strains based on 16S rRNA gene sequences corresponded to wider groups of species in terms of morphology. The overall secondary metabolite and fatty acid profiles, however, were not correlated to each other and neither were they correlated to the 16S rRNA phylogeny nor the morphology of the strains. Nevertheless, a minor part of the detected secondary metabolites (19% of all compounds) was present only in close relatives and can be thus considered as autapomorphic features.