From genome mining to phenotypic microarrays: Planctomycetes as source for novel bioactive molecules

Most members of the phylum Planctomycetes share many unusual traits that are unique for bacteria, since they divide independent of FtsZ through asymmetric budding, possess a complex life cycle and comprise a compartmentalized cell plan. Besides their complex cell biological features Planctomycetes are environmentally important and play major roles in global matter fluxes. Such features have been successfully employed in biotechnological applications such as the anaerobic oxidation of ammonium in wastewater treatment plants or the utilization of enzymes for biotechnological processes. However, little is known about planctomycetal secondary metabolites. This is surprising as Planctomycetes have several key features in common with known producers of small bioactive molecules such as Streptomycetes or Myxobacteria: a complex life style and large genome sizes. Planctomycetal genomes with an average size of 6.9 MB appear as tempting targets for drug discovery approaches. To enable the hunt for bioactive molecules from Planctomycetes, we performed a comprehensive genome mining approach employing the antiSMASH secondary metabolite identification pipeline and found 102 candidate genes or clusters within the analyzed 13 genomes. However, as most genes and operons related to secondary metabolite production are exclusively expressed under certain environmental conditions, we optimized Phenotype MicroArray protocols for Rhodopirellula baltica and Planctomyces limnophilus to allow high throughput screening of putative stimulating carbon sources. Our results point towards a previously postulated relationship of Planctomycetes with algae or plants, which secrete compounds that might serve as trigger to stimulate the secondary metabolite production in Planctomycetes. Thus, this study provides the necessary starting point to explore planctomycetal small molecules for drug development.     

Phylogenetic and Metabolic Diversity of Planctomycetes from Anaerobic, Sulfide- and Sulfur-Rich Zodletone Spring, Oklahoma

We investigated the phylogenetic diversity and metabolic capabilities of members of the phylum Planctomycetes in the anaerobic, sulfide-saturated sediments of a mesophilic spring (Zodletone Spring) in southwestern Oklahoma. Culture-independent analyses of 16S rRNA gene sequences generated using Planctomycetes-biased primer pairs suggested that an extremely diverse community of Planctomycetes is present at the spring. Although sequences that are phylogenetically affiliated with cultured heterotrophic Planctomycetes were identified, the majority of the sequences belonged to several globally distributed, as-yet-uncultured Planctomycetes lineages. Using complex organic media (aqueous extracts of the spring sediments and rumen fluid), we isolated two novel strains that belonged to the Pirellula-Rhodopirellula-Blastopirellula clade within the Planctomycetes. The two strains had identical 16S rRNA gene sequences, and their closest relatives were isolates from Kiel Fjord (Germany), Keauhou Beach (HI), a marine aquarium, and tissues of marine organisms (Aplysina sp. sponges and postlarvae of the giant tiger prawn Penaeus monodon). The closest recognized cultured relative of strain Zi62 was Blastopirellula marina (93.9% sequence similarity). Detailed characterization of strain Zi62 revealed its ability to reduce elemental sulfur to sulfide under anaerobic conditions, as well as its ability to produce acids from sugars; both characteristics may potentially allow strain Zi62 to survive and grow in the anaerobic, sulfide- and sulfur-rich environment at the spring source. Overall, this work indicates that anaerobic metabolic abilities are widely distributed among all major Planctomycetes lineages and suggests carbohydrate fermentation and sulfur reduction as possible mechanisms employed by heterotrophic Planctomycetes for growth and survival under anaerobic conditions.     

Planctomycetes in Lakes: Poor or Strong Competitors for Phosphorus?

Experiments were conducted with water samples from two perialpine lakes with differing eutrophication status in order to examine the effects of inorganic-nutrient amendments (nitrogen as NO₃⁻ or NH₄⁺ and phosphorus as PO₄³⁻) on the dynamics, structure, and composition of Planctomycetes and to test the hypothesis that the community structure of Planctomycetes members and that of the other bacteria (without Planctomycetes, here referred to as bacteria-wP, the most represented groups within the community) would be similarly impacted by nutrient additions. Initial samples were characterized by high total nitrogen-to-total phosphorus ratios (range, 39 to 55), suggesting P rather than N was the limiting nutrient for microbial communities. Consistent with this, P additions stimulated phytoplankton growth and affected the community structure of bacteria-wP but, surprisingly, not that of Planctomycetes. N additions did not significantly affect the community structures of bacteria-wP and Planctomycetes or the Planctomycetes phylotype composition. The estimated generation time of Planctomycetes was 123 h. These findings could suggest that the generally well-accepted statement that bacteria (as a whole) are superior to phytoplankton in the ability to obtain phosphorus under P limitation might actually not hold for Planctomycetes. Planctomycetes might be poor competitors for P that do not respond quickly to the nutrient supply, which may help explain why their abundance is low in aquatic systems. The alternative view that Planctomycetes could be strong competitors for P (storing it) is also discussed. Our findings highlight the need for further studies examining Planctomycetes-phosphorus relationships in aquatic ecosystems.     

Two overlooked species of Guzmania (Bromeliaceae) of the species-complex Massangea from Central America

Two new speciesGuzmania herrerae andG. scandens, that have been mistakenly identified asG. dissitiflora are described and illustrated. All three taxa belong to a natural complex of species that corresponds to the formerly recognized segregate genusMassangea E. Morren. However, without additional morphological and molecular evidence we believe it is premature to recognize this species complex as a genus separate fromGuzmania.     

Potential Conservation of Circadian Clock Proteins in the phylum Nematoda as Revealed by Bioinformatic Searches

Although several circadian rhythms have been described in C. elegans, its molecular clock remains elusive. In this work we employed a novel bioinformatic approach, applying probabilistic methodologies, to search for circadian clock proteins of several of the best studied circadian model organisms of different taxa (Mus musculus, Drosophila melanogaster, Neurospora crassa, Arabidopsis thaliana and Synechoccocus elongatus) in the proteomes of C. elegans and other members of the phylum Nematoda. With this approach we found that the Nematoda contain proteins most related to the core and accessory proteins of the insect and mammalian clocks, which provide new insights into the nematode clock and the evolution of the circadian system.     

Identification of Proteins Likely To Be Involved in Morphogenesis,Cell Division,and Signal Transduction in Planctomycetes by Comparative Genomics

Members of the Planctomycetes clade share many unusual features for bacteria. Their cytoplasm contains membrane-bound compartments, they lack peptidoglycan and FtsZ, they divide by polar budding, and they are capable of endocytosis. Planctomycete genomes have remained enigmatic, generally being quite large (up to 9 Mb), and on average, 55% of their predicted proteins are of unknown function. Importantly, proteins related to the unusual traits of Planctomycetes remain largely unknown. Thus, we embarked on bioinformatic analyses of these genomes in an effort to predict proteins that are likely to be involved in compartmentalization, cell division, and signal transduction. We used three complementary strategies. First, we defined the Planctomycetes core genome and subtracted genes of well-studied model organisms. Second, we analyzed the gene content and synteny of morphogenesis and cell division genes and combined both methods using a “guilt-by-association” approach. Third, we identified signal transduction systems as well as sigma factors. These analyses provide a manageable list of candidate genes for future genetic studies and provide evidence for complex signaling in the Planctomycetes akin to that observed for bacteria with complex life-styles, such as Myxococcus xanthus.     

A molecular phylogeny of bivalve mollusks: ancient radiations and divergences as revealed by mitochondrial genes

Background

Bivalves are very ancient and successful conchiferan mollusks (both in terms of species number and geographical distribution). Despite their importance in marine biota, their deep phylogenetic relationships were scarcely investigated from a molecular perspective, whereas much valuable work has been done on taxonomy, as well as phylogeny, of lower taxa.

Methodology/Principal Findings

Here we present a class-level bivalve phylogeny with a broad sample of 122 ingroup taxa, using four mitochondrial markers (MT-RNR1, MT-RNR2, MT-CO1, MT-CYB). Rigorous techniques have been exploited to set up the dataset, analyze phylogenetic signal, and infer a single final tree. In this study, we show the basal position of Opponobranchia to all Autobranchia, as well as of Palaeoheterodonta to the remaining Autobranchia, which we here propose to call Amarsipobranchia. Anomalodesmata were retrieved as monophyletic and basal to (Heterodonta + Pteriomorphia).

Conclusions/Significance

Bivalve morphological characters were traced onto the phylogenetic trees obtained from the molecular analysis; our analysis suggests that eulamellibranch gills and heterodont hinge are ancestral characters for all Autobranchia. This conclusion would entail a re-evaluation of bivalve symplesiomorphies.     

Whole-Genome Reciprocal BLAST Analysis Reveals that Planctomycetes Do Not Share an Unusually Large Number of Genes with Eukarya and Archaea

The genome sequences of Rhodopirellula baltica, formerly Pirellula sp. strain 1, Blastopirellula marina, Gemmata obscuriglobus, and Kuenenia stuttgartiensis were used in a series of pairwise reciprocal best-hit analyses to evaluate the contested evolutionary position of Planctomycetes. Contrary to previous reports which suggested that R. baltica had a high percentage of genes with closest matches to Archaea and Eukarya, we show here that these Planctomycetes do not share an unusually large number of genes with the Archaea or Eukarya, compared with other Bacteria. Thus, best-hit analyses may assign phylogenetic affinities incorrectly if close relatives are absent from the sequence database.     

South African fossil Cercopithecoidea: A re-assessment including a description of new material from Makapansgat,Sterkfontein and Taung

The fossil cercopithecoid material from South Africa has been reviewed according to sites and species. The 722 specimens considered comprise 6 genera including 16 taxa and come from 16 sites. Aspects of taxonomic controversy and interest are discussed. In particular, the Parapapio material from Makapansgat has been re-evaluated and the taxonomy of the genus Simopithecus is reconsidered. A number of proposals are put forward. Four new specimens from Makapansgat and one from Sterkfontein are described; a previously partially described specimen from Taung is re-described in detail.     

Systématique du groupe « Palaeochelys sensu lato – Mauremys » (Chelonii,Testudinoidea) du Tertiaire d’Europe occidentale : principaux résultats

Among fossil chelonians from the Tertiary of Western Europe, freshwater Testudinoidea (sensu Hirayama, 1985, Gaffney et Meylan, 1988) are abundant, and more particularly the “Palaeochelys sensu lato—Mauremys” group. This group has never been studied as a whole. Taxa of this group were before referred to extinct genera such as Ocadia, Clemmys, Emys or to the fossil genera Palaeochelys, Borkenia and Palaeoemys. These taxa are redefined, new taxa are described and a hypothesis of phylogenetic relationships is given.     

A case of mistaken identity,Opuntia abjecta,long-lost in synonymy under the Caribbean species,O. triacantha,and a reassessment of the enigmatic O. cubensis

Opuntia abjecta and O. militaris have been placed in synonymy under the Caribbean species O. triacantha for the past 30 years. Recent molecular phylogenetic evidence has shown, however, that O. abjecta and O. triacantha are actually in two very different clades suggesting that the Floridian endemic O. abjecta should be recognized as a distinct species. Here, we summarize major morphological differences between O. abjecta and O. triacantha. We also include new sequence data from the rare Cuban taxon, O. militaris, in a molecular phylogenetic analysis to determine its relationship to O. triacantha and O. abjecta. We discuss the putative hybrid taxa O. cubensis and O. ochrocentra, which currently are treated as synonyms. We also show through analysis of morphological and molecular characters that these two taxa were derived from two independent origins from divergent maternal progenitors, confirming that O. ochrocentra should not be treated as synonymous with O. cubensis. A key is provided for identifying these taxonomically confusing taxa and their close relatives. This study emphasizes the distinctions among O. abjecta, O. militaris, and O. triacantha and illustrates that extreme caution must be employed when using herbarium specimens for identifying species of Opuntia. It also indicates that broad phytogeographic assumptions regarding species’ relationships in Opuntia may sometimes be misleading. Hybridization and polyploidy are common in Opuntia and have played a role in the formation of new species in this group as well. A neotype is here designated for O. triacantha.     

Metagenomic Identification of Bacterioplankton Taxa and Pathways Involved in Microcystin Degradation in Lake Erie

Cyanobacterial harmful blooms (CyanoHABs) that produce microcystins are appearing in an increasing number of freshwater ecosystems worldwide, damaging quality of water for use by human and aquatic life. Heterotrophic bacteria assemblages are thought to be important in transforming and detoxifying microcystins in natural environments. However, little is known about their taxonomic composition or pathways involved in the process. To address this knowledge gap, we compared the metagenomes of Lake Erie free-living bacterioplankton assemblages in laboratory microcosms amended with microcystins relative to unamended controls. A diverse array of bacterial phyla were responsive to elevated supply of microcystins, including Acidobacteria, Actinobacteria, Bacteroidetes, Planctomycetes, Proteobacteria of the alpha, beta, gamma, delta and epsilon subdivisions and Verrucomicrobia. At more detailed taxonomic levels, Methylophilales (mainly in genus Methylotenera) and Burkholderiales (mainly in genera Bordetella, Burkholderia, Cupriavidus, Polaromonas, Ralstonia, Polynucleobacter and Variovorax) of Betaproteobacteria were suggested to be more important in microcystin degradation than Sphingomonadales of Alphaproteobacteria. The latter taxa were previously thought to be major microcystin degraders. Homologs to known microcystin-degrading genes (mlr) were not overrepresented in microcystin-amended metagenomes, indicating that Lake Erie bacterioplankton might employ alternative genes and/or pathways in microcystin degradation. Genes for xenobiotic metabolism were overrepresented in microcystin-amended microcosms, suggesting they are important in bacterial degradation of microcystin, a phenomenon that has been identified previously only in eukaryotic systems.     

Taxonomic Boundaries and Craniometric Variation in the Treeshrews (Scandentia,Tupaiidae) from the Palawan Faunal Region

The taxonomy of treeshrews (Order Scandentia) has long been complicated by ambiguous morphological species boundaries, and the treeshrews of the Palawan faunal region of the Philippines are no exception. Four named forms in the genus Tupaia Raffles, 1821, have been described from four island groups based on subtle qualitative morphological characters, and as many as three distinct species have been recognized. A recent molecular phylogenetic study of relationships among Tupaia species suggests that the two currently-recognized treeshrew species from the Palawan faunal region diverged very recently relative to other sister-species divergences within the genus and may not represent species-level taxonomic entities. Here we review the taxonomic and biogeographic histories of the Tupaia taxa from this region. We also collected craniodental data from 133 skulls of all four named forms, representing five island populations, and conducted univariate and multivariate analyses on these data. Our morphometric results are consistent with molecular results, further suggesting that there is insufficient evidence to recognize T. moellendorffi Matschie, 1898, as a separate species from T. palawanensis Thomas, 1894. Our analyses also revealed a craniodentally divergent population from the island of Balabac, which has never been considered a distinct subspecies (or species) from the population on Palawan. These results have conservation implications for the island populations in our analyses, but additional surveys and molecular evidence will be required to fully assess conservation priorities for the treeshrews of the Palawan faunal region.     

Characterization of the first planctomycetal outer membrane protein identifies a channel in the outer membrane of the anammox bacterium Kuenenia stuttgartiensis

Planctomycetes are a bacterial phylum known for their complex intracellular compartmentalization. While most Planctomycetes have two compartments, the anaerobic ammonium oxidizing (anammox) bacteria contain three membrane-enclosed compartments. In contrast to a long-standing consensus, recent insights suggested the outermost Planctomycete membrane to be similar to a Gram-negative outer membrane (OM). One characteristic component that differentiates OMs from cytoplasmic membranes (CMs) is the presence of outer membrane proteins (OMPs) featuring a β-barrel structure that facilitates passage of molecules through the OM. Although proteomic and genomic evidence suggested the presence of OMPs in several Planctomycetes, no experimental verification existed of the pore-forming function and localization of these proteins in the outermost membrane of these exceptional microorganisms. Here, we show via lipid bilayer assays that at least two typical OMP-like channel-forming proteins are present in membrane preparations of the anammox bacterium Kuenenia stuttgartiensis. One of these channel-forming proteins, the highly abundant putative OMP Kustd1878, was purified to homogeneity. Analysis of the channel characteristics via lipid bilayer assays showed that Kustd1878 forms a moderately cation-selective channel with a high current noise and an average single-channel conductance of about 170–190 pS in 1 M KCl. Antibodies were raised against the purified protein and immunogold localization indicated Kustd1878 to be present in the outermost membrane. Therefore, this work clearly demonstrates the presence of OMPs in anammox Planctomycetes and thus firmly adds to the emerging view that Planctomycetes have a Gram-negative cell envelope.     

rpoB gene as a novel molecular marker to infer phylogeny in Planctomycetales

The 16S rRNA gene has been used in the last decades as a gold standard for determining the phylogenetic position of bacteria and their taxonomy. It is a well conserved gene, with some variations, present in all bacteria and allows the reconstruction of genealogies of microorganisms. Nevertheless, this gene has its limitations when inferring phylogenetic relationships between closely related isolates. To overcome this problem, DNA–DNA hybridization appeared as a solution to clarify interspecies relationships when the sequence similarity of the 16S rRNA gene is above 97 %. However, this technique is time consuming, expensive and laborious and so, researchers developed other molecular markers such as sequencing of housekeeping or functional genes for accurate determination of bacterial phylogeny. One of these genes that have been used successfully, particularly in clinical microbiology, codes for the beta subunit of the RNA polymerase (rpoB). The rpoB gene is sufficiently conserved to be used as a molecular clock, it is present in all bacteria and it is a mono-copy gene. In this study, rpoB gene sequencing was applied to the phylum Planctomycetes. Based on the genomes of 19 planctomycetes it was possible to determine the correlation between the rpoB gene sequence and the phylogenetic position of the organisms at a 95–96 % sequence similarity threshold for a novel species. A 1200-bp fragment of the rpoB gene was amplified from several new planctomycetal isolates and their intra and inter-species relationships to other members of this group were determined based on a 96.3 % species border and 98.2 % for intraspecies resolution.