Cell surface proteome of the marine planctomycete Rhodopirellula baltica

The surface proteome (surfaceome) of the marine planctomycete Rhodopirellula baltica SH1(T) was studied using a biotinylation and a proteinase K approach combined with SDS-PAGE and mass spectrometry. 52 of the proteins identified in both approaches could be assigned to the group of potential surface proteins. Among them are some high molecular weight proteins, potentially involved in cell-cell attachment, that contain domains shown before to be typical for surface proteins like cadherin/dockerin domains, a bacterial adhesion domain or the fasciclin domain. The identification of proteins with enzymatic functions in the R. baltica surfaceome provides further clues for the suggestion that some degradative enzymes may be anchored onto the cell surface. YTV proteins, which have been earlier supposed to be components of the proteinaceous cell wall of R. baltica, were detected in the surface proteome. Additionally, 8 proteins with a novel protein structure combining a conserved type IV pilin/N-methylation domain and a planctomycete-typical DUF1559 domain were identified.     

Isolation and diversity of planctomycetes from the sponge Niphates sp., seawater, and sediment of Moreton Bay, Australia

Planctomycetes are ubiquitous in marine environment and were reported to occur in association with multicellular eukaryotic organisms such as marine macroalgae and invertebrates. Here, we investigate planctomycetes associated with the marine sponge Niphates sp. from the sub-tropical Australian coast by assessing their diversity using culture-dependent and -independent approaches based on the 16S rRNA gene. The culture-dependent approach resulted in the isolation of a large collection of diverse planctomycetes including some novel lineages of Planctomycetes from the sponge as well as sediment and seawater of Moreton Bay where this sponge occurs. The characterization of these novel planctomycetes revealed that cells of one unique strain do not possess condensed nucleoids, a phenotype distinct from other planctomycetes. In addition, a culture-independent clone library approach identified unique planctomycete 16S rRNA gene sequences closely related to other sponge-derived sequences. The analysis of tissue of the sponge Niphates sp. showed that the mesohyl of the sponge is almost devoid of microbial cells, indicating this species is in the group of ‘low microbial abundant’ (LMA) sponges. The unique planctomycete 16S rRNA gene sequences identified in this study were phylogenetically closely related to sequences from LMA sponges in other published studies. This study has revealed new insights into the diversity of planctomycetes in the marine environment and the association of planctomycetes with marine sponges.     

Cell compartmentalisation in planctomycetes: novel types of structural organisation for the bacterial cell

The organisation of cells of the planctomycete species Pirellula marina, Isosphaera pallida, Gemmata obscuriglobus, Planctomyces maris and "Candidatus Brocadia anammoxidans" was investigated based on ultrastructure derived from thin-sections of cryosubstituted cells, freeze-fracture replicas, and in the case of Gemmata obscuriglobus and Pirellula marina, computer-aided 3-D reconstructions from serial sections of cryosubstituted cells. All planctomycete cells display a peripheral ribosome-free region, termed here the paryphoplasm, surrounding the perimeter of the cell, and an interior region including any nucleoid regions as well as ribosome-like particles, bounded by a single intracytoplasmic membrane (ICM), and termed the pirellulosome in Pirellula species. Immunogold labelling and RNase-gold cytochemistry indicates that in planctomycetes all the cell DNA is contained wholly within the interior region bounded by the ICM, and the paryphoplasm contains no DNA but at least some of the cell's RNA. The ICM in Isosphaera pallida and Planctomyces maris is invaginated such that the paryphoplasm forms a major portion of the cell interior in sections, but in other planctomycetes it remains as a peripheral zone. In the anaerobic ammonium-oxidising ("anammox" process) chemoautotroph "Candidatus Brocadia anammoxidans" the interior region bounded by ICM contains a further internal single-membrane-bounded region, the anammoxosome. In Gemmata obscuriglobus, the interior ICM-bounded region contains the nuclear body, a double-membrane-bounded region containing the cell's nucleoid and all genomic DNA in addition to some RNA. Shared features of cell compartmentalisation in different planctomycetes are consistent with the monophyletic nature of the planctomycetes as a distinct division of the Bacteria. The shared organisational plan for the planctomycete cell constitutes a new type not known in cells of other bacteria.     

Detection of representatives of the Planctomycetes in Sphagnum peat bogs by molecular and cultivation methods

By means of fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes (FISH), it has been shown that members of the phylum Planctomycetes represent a numerically significant bacterial group in boreal Sphagnum peat bogs. The population size of planctomycetes in oxic layers of the peat bog profile was in the range of 0.4-2.0 x 10(7) cells per g of wet peat, comprising 4 to 13% of the total bacterial cell number. A novel effective approach that combined a traditional cultivation technique with FISH-mediated monitoring of the target organism during the isolation procedure has been developed for the isolation of planctomycetes. Using this approach, we succeeded in isolating several peat-inhabiting planctomycetes in a pure culture. Sequencing of the 16S rRNA genes from two of these isolates, strains A10 and MPL7, showed that they belonged to the planctomycete lineages defined by the genera Gemmata and Planctomyces, respectively. The 16S rRNA gene sequence similarity between strains A10 and MPL7 and the phylogenetically closest organisms, namely, Gemmata obscuriglobus and Planctomyces limnophilus, was only 90%. These results suggest that the indigenous planctomycetes inhabiting Sphagnum peat bogs are so far unknown organisms.     

Detection of representatives of the Planctomycetes in Sphagnum peat bogs by molecular and cultivation approaches

By means of fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes (FISH), it has been shown that members of the phylum Planctomycetes represent a numerically significant bacterial group in boreal Sphagnum peat bogs. The population size of planctomycetes in oxic layers of the peat bog profile was in the range of 0.4–2.0 × 10⁷ cells per g of wet peat, comprising 4 to 13% of the total bacterial cell number. A novel effective approach that combined a traditional cultivation technique with FISH-mediated monitoring of the target organism during the isolation procedure has been developed for the isolation of planctomycetes. Using this approach, we succeeded in isolating several peat-inhabiting planctomycetes in a pure culture. Sequencing of the 16S rRNA genes from two of these isolates, strains A10 and MPL7, showed that they belonged to the planctomycete lineages defined by the genera Gemmata and Planctomyces, respectively. The 16S rRNA gene sequence similarity between strains A10 and MPL7 and the phylogenetically closest organisms, namely, Gemmata obscuriglobus and Planctomyces limnophilus, was only 90%. These results suggest that the indigenous planctomycetes inhabiting Sphagnum peat bogs are so far unknown organisms.     

Extracting sequence motifs and the phylogenetic features of SNARE-dependent membrane traffic

The SNARE proteins are required for membrane fusion during intracellular vesicular transport and for its specificity. Only the unique combination of SNARE proteins (cognates) can be bound and can lead to membrane fusion, although the characteristics of the possible specificity of the binding combinations encoded in the SNARE sequences have not yet been determined. We discovered by whole genome sequence analysis that sequence motifs (conserved sequences) in the SNARE motif domains for each protein group correspond to localization sites or transport pathways. We claim that these motifs reflect the specificity of the binding combinations of SNARE motif domains. Using these motifs, we could classify SNARE proteins from 48 organisms into their localization sites or transport pathways. The classification result shows that more than 10 SNARE subgroups are kingdom specific and that the SNARE paralogs involved in the plasma membrane-related transport pathways have developed greater variations in higher animals and higher plants than those involved in the endoplasmic reticulum-related transport pathways throughout eukaryotic evolution.     

Sequence analysis of the non-recurring C-terminal domains shows that insect lipoprotein receptors constitute a distinct group of LDL receptor family members

Lipoprotein-mediated delivery of lipids in mammals involves endocytic receptors of the low density lipoprotein (LDL) receptor (LDLR) family. In contrast, in insects, the lipoprotein, lipophorin (Lp), functions as a reusable lipid shuttle in lipid delivery, and these animals, therefore, were not supposed to use endocytic receptors. However, recent data indicate additional endocytic uptake of Lp, mediated by a Lp receptor (LpR) of the LDLR family. The two N-terminal domains of LDLR family members are involved in ligand binding and dissociation, respectively, and are composed of a mosaic of multiple repeats. The three C-terminal domains, viz., the optional O-linked glycosylation domain, the transmembrane domain, and the intracellular domain, are of a non-repetitive sequence. The present classification of newly discovered LDLR family members, including the LpRs, bears no relevance to physiological function. Therefore, as a novel approach, the C-terminal domains of LDLR family members across the entire animal kingdom were used to perform a sequence comparison analysis in combination with a phylogenetic tree analysis. The LpRs appeared to segregate into a specific group distinct from the groups encompassing the other family members, and each of the three C-terminal domains of the insect receptors is composed of unique set of sequence motifs. Based on conservation of sequence motifs and organization of these motifs in the domains, LpR resembles most the groups of the LDLRs, very low density lipoprotein (VLDL) receptors, and vitellogenin receptors. However, in sequence aspects in which LpR deviates from these three receptor groups, it most notably resembles LDLR-related protein-2, or megalin. These features might explain the functional differences disclosed between insect and mammalian lipoprotein receptors.     

Candidatus "Scalindua brodae", sp. nov., Candidatus "Scalindua wagneri", sp. nov., two new species of anaerobic ammonium oxidizing bacteria

Anaerobic ammonium oxidation (anammox) is both a promising process in wastewater treatment and a long overlooked microbial physiology that can contribute significantly to biological nitrogen cycling in the world's oceans. Anammox is mediated by a monophyletic group of bacteria that branches deeply in the Planctomycetales. Here we describe a new genus and species of anaerobic ammonium oxidizing planctomycetes, discovered in a wastewater treatment plant (wwtp) treating landfill leachate in Pitsea, UK. The biomass from this wwtp showed high anammox activity (5.0 +/- 0.5 nmol/mg protein/min) and produced hydrazine from hydroxylamine, one of the unique features of anammox bacteria. Eight new planctomycete 16S rRNA gene sequences were present in the 16S rRNA gene clone library generated from the biomass. Four of these were affiliated to known anammox 16S rRNA gene sequences, but branched much closer to the root of the planctomycete line of descent. Fluorescence in situ hybridization (FISH) with oligonucleotide probes specific for these new sequences showed that two species (belonging to the same genus) together made up > 99% of the planctomycete population which constituted 20% of the total microbial community. The identification of these organisms as typical anammox bacteria was confirmed with electron microscopy and lipid analysis. The new species, provisionally named Candidatus "Scalindua brodae" and "Scalindua wagneri" considerably extend the biodiversity of the anammox lineage on the 16S rRNA gene level, but otherwise resemble known anammox bacteria. Simultaneously, another new species of the same genus, Candidatus "Scalindua sorokinii", was detected in the water column of the Black Sea, making this genus the most widespread of all anammox bacteria described so far.     

Calmodulin’s flexibility allows for promiscuity in its interactions with target proteins and peptides

The small bilobal calcium regulatory protein calmodulin (CaM) activates numerous target enzymes in response to transient changes in intracellular calcium concentrations. Binding of calcium to the two helix-loop-helix calcium-binding motifs in each of the globular domains induces conformational changes that expose a methionine-rich hydrophobic patch on the surface of each domain of the protein, which it uses to bind to peptide sequences in its target enzymes. Although these CaM-binding domains typically have little sequence identity, the positions of several bulky hydrophobic residues are often conserved, allowing for classification of CaM-binding domains into recognition motifs, such as the 1–14 and 1–10 motifs. For calcium-independent binding of CaM, a third motif known as the IQ motif is also common. Many CaM-peptide complexes have globular conformations, where CaM’s central linker connecting the two domains unwinds, allowing the protein to wrap around a single predominantly α-helical target peptide sequence. However, novel structures have recently been reported where the conformation of CaM is highly dissimilar to these globular complexes, in some instances with less than a full compliment of bound calcium ions, as well as novel stoichiometries. Furthermore, many divergent CaM isoforms from yeast and plant species have been discovered with unique calcium-binding and enzymatic activation characteristics compared to the single CaM isoform found in mammals.     

Functional plasticity of CH domains

With the refinement of algorithms for the identification of distinct motifs from sequence databases, especially those using secondary structure predictions, new protein modules have been determined in recent years. Calponin homology (CH) domains were identified in a variety of proteins ranging from actin cross-linking to signaling and have been proposed to function either as autonomous actin binding motifs or serve a regulatory function. Despite the overall structural conservation of the unique CH domain fold, the individual modules display a quite striking functional variability. Analysis of the actopaxin/parvin protein family suggests the existence of novel (type 4 and type 5) CH domain families which require special attention, as they appear to be a good example for how CH domains may function as scaffolds for other functional motifs of different properties.     

Molecular evolution of the equilibrative nucleoside transporter family: identification of novel family members in prokaryotes and eukaryotes

Equilibrative nucleoside transporters (ENTs) are integral membrane proteins which enable the movement of hydrophilic nucleosides and nucleoside analogs down their concentration gradients across cell membranes. ENTs were only recently characterized at the molecular level, and little is known about the tertiary structure or distribution of these proteins in nonmammalian organisms. To identify conserved regions, residues, and motifs of ENTs that may indicate functionally important parts of the protein and to better understand the evolutionary history of this protein family, we conducted an exhaustive analysis to characterize and compare ENTs in taxonomically diverse organisms. We have identified novel ENT family members in humans, mice, fish, tunicates, slime molds, and bacteria. This greatly extends our knowledge on the distribution of the ENTs in eukaryotes, and we have identified, for the first time, family members in bacteria. The prokaryote ENTs are attractive models for future studies on transporter tertiary structure and mechanism of substrate translocation. Using sequence similarities, we have identified regions, residues, and motifs that are conserved across all family members. These areas are presumably correlated with function and therefore are important targets for future analysis. Finally, we propose an evolutionary history for the ENT family which clarifies the origin(s) of multiple isoforms in different taxa.     

Isolation and molecular identification of planctomycete bacteria from postlarvae of the giant tiger prawn, Penaeus monodon.

Bacteria phenotypically resembling members of the phylogenetically distinct planctomycete group of the domain Bacteria were isolated from postlarvae of the giant tiger prawn, Penaeus monodon. A selective medium designed in the light of planctomycete antibiotic resistance characteristics was used for this isolation. Planctomycetes were isolated from both healthy and monodon baculovirus-infected prawn postlarvae. The predominant colony type recovered from postlarvae regardless of viral infection status was nonpigmented. Other, less commonly observed types were pink or orange pigmented. A planctomycete-specific 16S rRNA-directed probe was designed and used to screen the isolates for their identity as planctomycetes prior to molecular phylogenetic characterization. 16S rRNA genes from nine prawn isolates together with two planctomycete reference strains (Planctomyces brasiliensis and Gemmata obscuriglobus) were sequenced and compared with reference sequences from the planctomycetes and other members of the domain Bacteria. Phylogenetic analyses and sequence signatures of the 16S rRNA genes demonstrated that the prawn isolates were members of the planctomycete group. Five representatives of the predominant nonpigmented colony type were members of the Pirellula group within the planctomycetes, as were three pink-pigmented colony type representatives. Homology values and tree topology indicated that representatives of the nonpigmented and pink-pigmented colony types formed two discrete clusters within the Pirellula group, not identical to any known Pirellula species. A sole representative of the orange colony type was a member of the Planctomyces group, virtually identical in 16S rDNA sequence to P. brasiliensis, and exhibited distinctive morphology.     

Beyond the bacterium: planctomycetes challenge our concepts of microbial structure and function

Planctomycetes form a distinct phylum of the domain Bacteria and possess unusual features such as intracellular compartmentalization and a lack of peptidoglycan in their cell walls. Remarkably, cells of the genus Gemmata even contain a membrane-bound nucleoid analogous to the eukaryotic nucleus. Moreover, the so-called 'anammox' planctomycetes have a unique anaerobic, autotrophic metabolism that includes the ability to oxidize ammonium; this process is dependent on a characteristic membrane-bound cell compartment called the anammoxosome, which might be a functional analogue of the eukaryotic mitochondrion. The compartmentalization of planctomycetes challenges our hypotheses regarding the origins of eukaryotic organelles. Furthermore, the recent discovery of both an endocytosis-like ability and proteins homologous to eukaryotic clathrin in a planctomycete marks this phylum as one to watch for future research on the origin and evolution of the eukaryotic cell.     

Detailed proteome analysis of growing cells of the planctomycete Rhodopirellula baltica SH1T

Rhodopirellula baltica SH1(T), which was isolated from the water column of the Kieler Bight, a bay in the southwestern Baltic Sea, is a marine aerobic, heterotrophic representative of the ubiquitous bacterial phylum Planctomycetes. We analyzed the R. baltica proteome by applying different preanalytical protein as well as peptide separation techniques (1-D and 2-DE, HPLC separation) prior to MS. That way, we could identify a total of 1115 nonredundant proteins from the intracellular proteome and from different cell wall protein fractions. With the contribution of 709 novel proteins resulting from this study, the current comprehensive R. baltica proteomic dataset consists of 1267 unique proteins (accounting for 17.3% of the total putative protein-coding ORFs), including 261 proteins with a predicted signal peptide. The identified proteins were functionally categorized using Clusters of Orthologous Groups (COGs), and their potential cellular locations were predicted by bioinformatic tools. A unique protein family that contains several YTV domains and is rich in cysteine and proline was found to be a component of the R. baltica proteinaceous cell wall. Based on this comprehensive proteome analysis a global schema of the major metabolic pathways of growing R. baltica cells was deduced.     

Structure-based classification of 45 FK506-binding proteins

The FK506-binding proteins (FKBPs) are a unique group of chaperones found in a wide variety of organisms. They perform a number of cellular functions including protein folding, regulation of cytokines, transport of steroid receptor complexes, nucleic acid binding, histone assembly, and modulation of apoptosis. These functions are mediated by specific domains that adopt distinct tertiary conformations. Using the Threading/ASSEmbly/Refinement (TASSER) approach, tertiary structures were predicted for a total of 45 FKBPs in 23 species. These models were compared with previously characterized FKBP solution structures and the predicted structures were employed to identify groups of homologous proteins. The resulting classification may be utilized to infer functional roles of newly discovered FKBPs. The three-dimensional conformations revealed that this family may have undergone several modifications throughout evolution, including loss of N- and C-terminal regions, duplication of FKBP domains as well as insertions of entire functional motifs. Docking simulations suggest that additional sequence segments outside FKBP domains may modulate the binding affinity of FKBPs to immunosuppressive drugs. The docking models also indicate the presence of a helix-loop-helix (HLH) region within a subset of FKBPs, which may be responsible for the interaction between this group of proteins and nucleic acids.     

High abundance of planctomycetes in anoxic layers of a Sphagnum peat bog

The depth distribution of planctomycete abundance has been examined in six different sites of the Sphagnum peat bog in Bakchar, Tomsk oblast, Russia. In situ hybridization of peat with the fluorescently labeled oligonucleotide probes PLA46 and PLA886, reported to be group-specific for representatives of the phylum Planctomycetes, revealed two distinct population maxima of these bacteria in all of the profiles examined. The first population maximum was detected in the uppermost, oxic layer of the bog profile, while the second maximum was located at a depth of 30 cm below the water table level. The population sizes of planctomycetes in the uppermost layer and at a depth of 30 cm were of the same order of magnitude and comprised 0.5-1.5 x 10(7) and 0.4-0.7 x 10(7) cells per g of wet peat, respectively. Only 25-30% of the total number of planctomycete cells in the anoxic layer could be detected if the probe PLA886, whose target specificity is restricted to taxonomically characterized aerobic planctomycetes of the genera Gemmata, Planctomyces, Pirellula, and Isosphaera, was used alone. Other planctomycete cells in this layer were detected only with the probe PLA46, which possesses a much wider scope. This suggests the affiliation of these organisms with a yet undescribed phylogenetic subgroup within the Planctomycetes.     

The importance of conserved features of yeast actin-binding protein 1 (abp1p): the conditional nature of essentiality

Saccharomyces cerevisiae Actin-Binding Protein 1 (Abp1p) is a member of the Abp1 family of proteins, which are in diverse organisms including fungi, nematodes, flies, and mammals. All proteins in this family possess an N-terminal Actin Depolymerizing Factor Homology (ADF-H) domain, a central Proline-Rich Region (PRR), and a C-terminal SH3 domain. In this study, we employed sequence analysis to identify additional conserved features of the family, including sequences rich in proline, glutamic acid, serine, and threonine amino acids (PEST), which are found in all family members examined, and two motifs, Conserved Fungal Motifs 1 and 2 (CFM1 and CFM2), that are conserved in fungi. We also discovered that, similar to its mammalian homologs, Abp1p is phosphorylated in its PRR. This phosphorylation is mediated by the Cdc28p and Pho85p kinases, and it protects Abp1p from proteolysis mediated by the conserved PEST sequences. We provide evidence for an intramolecular interaction between the PRR region and SH3 domain that may be affected by phosphorylation. Although deletion of CFM1 alone caused no detectable phenotype in any genetic backgrounds or conditions tested, deletion of this motif resulted in a significant reduction of growth when it was combined with a deletion of the ADF-H domain. Importantly, this result demonstrates that deletion of highly conserved domains on its own may produce no phenotype unless the domains are assayed in conjunction with deletions of other functionally important elements within the same protein. Detection of this type of intragenic synthetic lethality provides an important approach for understanding the function of individual protein domains or motifs.