Ulrike Brandt

Actinorhodopsin genes discovered in diverse freshwater habitats and among cultivated freshwater Actinobacteria

Microbial rhodopsins are membrane proteins that utilize a retinal chromophore to harvest sunlight for energetic and photosensory functions. Recently, a group of novel rhodopsin sequences named ‘actinorhodopsins’ (ActRs) was hypothesized to exist among uncultured planktonic Actinobacteria. ActRs were discovered by mining metagenomic data obtained during the Venter Institutes Global Ocean Sampling expedition, from a hypersaline lagoon, two estuaries and a freshwater lake. On the basis of these findings, and many studies that show Actinobacteria are common inhabitants of lakes, we predicted that ActR genes would likely be present in other freshwater habitats and among the genomes of cultivated Actinobacteria. Using degenerate polymerase chain reaction primers, we discovered an ActR gene present in an actinobacterial isolate of the family Microbacteriaceae. Isolate MWH-Uga1 was cultivated prior to this study from a freshwater pond in Uganda and belongs to a group of Actinobacteria previously identified in freshwater ecosystems. ActR genes were also discovered present in numerous mixed cultures containing freshwater Actinobacteria and among environmental DNA samples obtained from three freshwater sources; a small woodland pond and the Laurentian Great Lakes Superior and Erie. An analysis of small subunit ribosomal RNA genes from metagenomic DNA samples harboring ActR genes suggests that organisms belonging to the acI lineage, an uncultured group of Actinobacteria commonly present in fresh waters, may utilize rhodopsins. The co-occurrence of an acI organism with a specific ActR variant in a mixed culture supports our hypothesis.

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.

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.

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.

Polynucleobacter cosmopolitanus sp. nov., free-living planktonic bacteria inhabiting freshwater lakes and rivers

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.

Limnohabitans australis sp. nov., isolated from a freshwater pond, and emended description of the genus Limnohabitans

A chemo-organotrophic, aerobic, non-motile strain, MWH-BRAZ-DAM2DT, isolated from a freshwater pond in Brazil, was characterized phenotypically, phylogenetically and chemotaxonomically. Phylogenetic analysis of 16S rRNA gene sequences indicated affiliation of the strain with the genus Limnohabitans (Comamonadaceae, Betaproteobacteria). 16S rRNA gene sequence similarities between the isolate and Limnohabitans curvus MWH-C5T, representing the type species of the genus, and the type strains of Limnohabitans parvus and Limnohabitans planktonicus were 98.2, 96.5 and 97.0 %, respectively. DNA–DNA reassociation analyses with DNA of the type strains of all three previously described Limnohabitans species revealed similarity values in the range 26.2–44.6 %. The predominant fatty acids of the isolate were C16 : 1ω7c/ω6c, C16 : 0, C12 : 0 and C8 : 0 3-OH, the major quinone was ubiquinone Q-8 and the DNA G+C content was 55.8 mol%. The isolate could be discriminated from the type strains of the three Limnohabitans species by several phenotypic traits including differences in the utilization of several carbon sources. Based on the phylogeny of the isolate and its differences from the three most closely related species, the isolate represents a novel species for which the name Limnohabitans australis sp. nov. is proposed. The type strain is MWH-BRAZ-DAM2DT (=DSM 21646T=CCUG 56719T).

Polynucleobacter acidiphobus sp. nov., a representative of an abundant group of planktonic freshwater bacteria.

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)ω7c (feature 3), C(18 : 1)ω7c 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)).

Polynucleobacter rarus sp. nov., a free-living planktonic bacterium isolated from an acidic lake

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)ω7c, 11-methyl C(18 : 1)ω7c, feature 3 (including C(16 : 1)ω7c), 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)ω8c 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)).

Dihydrolipoamide Dehydrogenases of Advenella mimigardefordensis and Ralstonia eutropha Catalyze Cleavage of 3,3′-Dithiodipropionic Acid into 3-Mercaptopropionic Acid

ABSTRACT The catabolism of the disulfide 3,3′-dithiodipropionic acid (DTDP) is initiated by the reduction of its disulfide bond. Three independent Tn5::mob-induced mutants of Advenella mimigardefordensis strain DPN7T were isolated that had lost the ability to utilize DTDP as the sole source of carbon and energy and that harbored the transposon insertions in three different sites of the same dihydrolipoamide dehydrogenase gene encoding the E3 subunit of the pyruvate dehydrogenase multi-enzyme complex of this bacterium (LpdAAm). LpdAAm was analyzed in silico and compared to homologous proteins, thereby revealing high similarities to the orthologue in Ralstonia eutropha H16 (PdhLRe). Both bacteria are able to cleave DTDP into two molecules of 3-mercaptopropionic acid (3MP). A. mimigardefordensis DPN7T converted 3MP to 3-sulfinopropionic acid, whereas R. eutropha H16 showed no growth with DTDP as the sole carbon source but was instead capable of synthesizing heteropolythioesters using the resulting cleavage product 3MP. Subsequently, the genes lpdAAm and pdhLRe were cloned, heterologously expressed in Escherichia coli applying the pET23a expression system, purified, and assayed by monitoring the oxidation of NADH. The physiological substrate lipoamide was reduced to dihydrolipoamide with specific activities of 1,833 mkat/kg of protein (LpdAAm) or 1,667 mkat/kg of protein (PdhLRe). Reduction of DTDP was also unequivocally detected with the purified enzymes, although the specific enzyme activities were much lower: 0.7 and 0.5 mkat/kg protein, respectively.

Versatile metabolic adaptations of Ralstonia eutropha H16 to a loss of PdhL, the E3 component of the pyruvate dehydrogenase complex.

ABSTRACT A previous study reported that the Tn5-induced poly(3-hydroxybutyric acid) (PHB)-leaky mutant Ralstonia eutropha H1482 showed a reduced PHB synthesis rate and significantly lower dihydrolipoamide dehydrogenase (DHLDH) activity than the wild-type R. eutropha H16 but similar growth behavior. Insertion of Tn5 was localized in the pdhL gene encoding the DHLDH (E3 component) of the pyruvate dehydrogenase complex (PDHC). Taking advantage of the available genome sequence of R. eutropha H16, observations were verified and further detailed analyses and experiments were done. In silico genome analysis revealed that R. eutropha possesses all five known types of 2-oxoacid multienzyme complexes and five DHLDH-coding genes. Of these DHLDHs, only PdhL harbors an amino-terminal lipoyl domain. Furthermore, insertion of Tn5 in pdhL of mutant H1482 disrupted the carboxy-terminal dimerization domain, thereby causing synthesis of a truncated PdhL lacking this essential region, obviously leading to an inactive enzyme. The defined ΔpdhL deletion mutant of R. eutropha exhibited the same phenotype as the Tn5 mutant H1482; this excludes polar effects as the cause of the phenotype of the Tn5 mutant H1482. However, insertion of Tn5 or deletion of pdhL decreases DHLDH activity, probably negatively affecting PDHC activity, causing the mutant phenotype. Moreover, complementation experiments showed that different plasmid-encoded E3 components of R. eutropha H16 or of other bacteria, like Burkholderia cepacia, were able to restore the wild-type phenotype at least partially. Interestingly, the E3 component of B. cepacia possesses an amino-terminal lipoyl domain, like the wild-type H16. A comparison of the proteomes of the wild-type H16 and of the mutant H1482 revealed striking differences and allowed us to reconstruct at least partially the impressive adaptations of R. eutropha H1482 to the loss of PdhL on the cellular level.