Evolutionary Remodeling of the Cell Envelope in Bacteria of the Planctomycetes Phylum.

Bacteria of the Planctomycetes phylum have many unique cellular features, such as extensive membrane invaginations and the ability to import macromolecules. These features raise intriguing questions about the composition of their cell envelopes. In this study, we have used microscopy, phylogenomics, and proteomics to examine the composition and evolution of cell envelope proteins in Tuwongella immobilis and other members of the Planctomycetes. Cryo-electron tomography data indicated a distance of 45 nm between the inner and outer membranes in T. immobilis. Consistent with the wide periplasmic space, our bioinformatics studies showed that the periplasmic segments of outer-membrane proteins in type II secretion systems are extended in bacteria of the order Planctomycetales. Homologs of two highly abundant cysteine-rich cell wall proteins in T. immobilis were identified in all members of the Planctomycetales, whereas genes for peptidoglycan biosynthesis and cell elongation have been lost in many members of this bacterial group. The cell wall proteins contain multiple copies of the YTV motif, which is the only domain that is conserved and unique to the Planctomycetales. Earlier diverging taxa in the Planctomycetes phylum contain genes for peptidoglycan biosynthesis but no homologs to the YTV cell wall proteins. The major remodeling of the cell envelope in the ancestor of the Planctomycetales coincided with the emergence of budding and other unique cellular phenotypes. The results have implications for hypotheses about the process whereby complex cellular features evolve in bacteria.

Alienimonas chondri sp. nov., a novel planctomycete isolated from the biofilm of the red alga Chondrus crispus.

The phylum Planctomycetes comprises bacteria with peculiar and very unique characteristics among prokaryotes. In marine environments, macroalgae biofilms are well known for harboring planctomycetal diversity. Here, we describe a novel isolate obtained from the biofilm of the red alga Chondrus crispus collected at a rocky beach in Porto, Portugal. The novel strain LzC2T is motile, rosette-forming with spherical- to ovoid-shaped cells. LzC2T forms magenta- to pinkish-colored colonies in M13 and M14 media. Transmission and scanning electron microscopy observations showed a division by polar and lateral budding. Mother cells are connected to the daughter cells by a tubular neck-like structure. The strain requires salt for growth. Vitamins are not required for growth. Optimal growth occurs from 15 to 30°C and within a pH range from 5.5 to 10.0. Major fatty acids are anteiso-C15:0 (54.2%) and iso-C15:0 (19.5%). Phosphatidylglycerol, diphosphatidylglycerol and an unidentified glycolipid represent the main lipids and menaquinone 6 (MK-6) is the only quinone present. 16S rRNA gene-based phylogenetic analysis supports the affiliation to the phylum Planctomycetes and family Planctomycetaceae, with Alienimonas as the closest relative. Strain LzC2T shares 97% 16S rRNA gene sequence similarity with Alienimonas californiensis. LzC2T has a genome size of 5.3 Mb and a G+C content of 68.3%. Genotypic and phenotypic comparison with the closest relatives strongly suggest that LzC2T (=CECT 30038T=LMG XXXT) is a new species of the genus Alienimonas, for which we propose the name Alienimonas chondri sp. nov., represented by LzC2T as type strain. 16S rRNA gene accession number: GenBank=MN757873.1. Genome accession number: GenBank=WTPX00000000.

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 ß-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 170-190pS in 1M 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.

Nuclear Pore-Like Structures in a Compartmentalized Bacterium.

Planctomycetes are distinguished from other Bacteria by compartmentalization of cells via internal membranes, interpretation of which has been subject to recent debate regarding potential relations to Gram-negative cell structure. In our interpretation of the available data, the planctomycete Gemmata obscuriglobus contains a nuclear body compartment, and thus possesses a type of cell organization with parallels to the eukaryote nucleus. Here we show that pore-like structures occur in internal membranes of G.obscuriglobus and that they have elements structurally similar to eukaryote nuclear pores, including a basket, ring-spoke structure, and eight-fold rotational symmetry. Bioinformatic analysis of proteomic data reveals that some of the G. obscuriglobus proteins associated with pore-containing membranes possess structural domains found in eukaryote nuclear pore complexes. Moreover, immunogold labelling demonstrates localization of one such protein, containing a ß-propeller domain, specifically to the G. obscuriglobus pore-like structures. Finding bacterial pores within internal cell membranes and with structural similarities to eukaryote nuclear pore complexes raises the dual possibilities of either hitherto undetected homology or stunning evolutionary convergence.

Anammox Biochemistry: a Tale of Heme c Proteins.

Anaerobic ammonium-oxidizing (anammox) bacteria are one of the latest scientific discoveries in the biogeochemical nitrogen cycle. These microorganisms are able to oxidize ammonium (NH4+) with nitrite (NO2-) as the oxidant instead of oxygen and form dinitrogen (N2) as the end product. Recent research has shed a light on the biochemistry underlying anammox metabolism with two key intermediates, nitric oxide (NO) and hydrazine (N2H4). Substrates and intermediates are converted exploiting the catalytic and electron-transfer potentials of c-type heme proteins known from numerous biochemical reactions and that have acquired new functionality in anammox biochemistry. On a global scale, anammox bacteria significantly contribute to the removal of fixed nitrogen from the environment and the process finds rapidly increasing interest in wastewater treatment.

Permanent draft genome of 'Rhodopirellula islandica' strain K833.

The planctomycete strain K833 was isolated from cold waters at the coast of Island and is tentatively named 'Rhodopirellula islandica'. It has a lower temperature range for growth than other genome-sequenced Rhodopirellula strains affiliating to Rhodopirellula baltica and 'Rhodopirellula europaea'. The permanent draft genome of strain K833 was obtained as part of a larger study on the biogeography of Rhodopirellula species in European marine waters. The genome consists of 55 contigs with a genome size of 7,433,200 bp. With an average nucleotide identity of 81% to related genomes of R. baltica and 'R. europaea' and more than 4000 common genes, it will be a valuable source for the study of temperature adaptation of planctomycete genomes.

[Candidatus "Jettenia moscovienalis" sp. nov., a New Species of Bacteria Carrying out Anaerobic Ammonium Oxidation].

A new species of bacteria oxidizing ammonium with nitrite under anoxic conditions was isolated from the activated sludge of a semi-industrial bioreactor treating digested sludge of the Kuryanovo wastewater treatment plant (Moscow, Russia). Physiological, morphological, and molecular genetic characterization of the isolate was carried out. The cells were ovoid (-0.5 x 0.8 µm), with the intracellular membrane structures characteristic of anammox bacteria (anammoxosome and paryphoplasm); unlike other anammox bacteria, it possessed extensive intracellular membrane structures located in layers parallel to the cytoplasmic membrane, but never close to the anammoxosome. The cells formed aggregates 5-28 µm in diameter and readily attached to solid surfaces. The cells were morphologically labile, easily plasmolyzed, and lost their content. Doubling time was 28 days, µ(max) = 0.025 day(-1); optimal temperature and pH for growth were 20-45 degrees C and 8.0, respectively. Phylogenetic analysis of the 16S rRNA gene sequences suggested its classification as a new species of the candidate genus Jettenia (order Planctomycetales). The name Candidatus "Jettenia moscovienalis" sp. nov. was proposed for the new bacterium.

Roseimaritima ulvae gen. nov., sp. nov. and Rubripirellula obstinata gen. nov., sp. nov. two novel planctomycetes isolated from the epiphytic community of macroalgae.

Four isolates, belonging to the deep-branching phylum Planctomycetes, were recovered from the biofilm of two marine macroalgae, Ulva sp. and Laminaria sp., from the Northern coast of Portugal. These strains were light pink- or red-pigmented; the cells were variable in shape and usually organized in rosettes. They had a dimorphic cell cycle with budding reproduction. The organisms were chemoheterotrophic, strictly aerobic and mesophilic. The 16S rRNA gene sequence analysis showed that the strains belong to the family Planctomycetaceae with Rhodopirellula as the closest genus. The isolates form two separate branches (strain LF1(T) forms one branch and the strains UC8(T), UF3 and UF42 form a second branch) clearly separated from Rhodopirellula baltica with 94.2% and 93.8% 16S rRNA gene sequence similarity, respectively. Based on differential characteristics that distinguish the novel genera from R. baltica, such as cell size and shape, ultrastructure, enzymatic activities, substrate utilization pattern, fatty acid composition, phospholipid profiles and phylogeny we propose that the isolates represent two novel genera of the order Planctomycetales, Roseimaritima ulvae gen. nov., sp. nov. (type strain is UC8(T)=DSM 25454(T)=LMG 27778(T)) and Rubripirellula obstinata gen. nov., sp. nov. (type strain is LF1(T)=LMG 27779(T)=CECT 8602(T)).

Structural studies of planctomycete Gemmata obscuriglobus support cell compartmentalisation in a bacterium.

Members of phylum Planctomycetes have been proposed to possess atypical cell organisation for the Bacteria, having a structure of sectioned cells consistent with internal compartments surrounded by membranes. Here via electron tomography we confirm the presence of compartments in the planctomycete Gemmata obscuriglobus cells. Resulting 3-D models for the most prominent structures, nuclear body and riboplasm, demonstrate their entirely membrane - enclosed nature. Immunogold localization of the FtsK protein also supports the internal organisation of G.obscuriglobus cells and their unique mechanism of cell division. We discuss how these new data expand our knowledge on bacterial cell biology and suggest evolutionary consequences of the findings.

Characterization of a planctomycetal organelle: a novel bacterial microcompartment for the aerobic degradation of plant saccharides.

Bacterial microcompartments (BMCs) are organelles that encapsulate functionally linked enzymes within a proteinaceous shell. The prototypical example is the carboxysome, which functions in carbon fixation in cyanobacteria and some chemoautotrophs. It is increasingly apparent that diverse heterotrophic bacteria contain BMCs that are involved in catabolic reactions, and many of the BMCs are predicted to have novel functions. However, most of these putative organelles have not been experimentally characterized. In this study, we sought to discover the function of a conserved BMC gene cluster encoded in the majority of the sequenced planctomycete genomes. This BMC is especially notable for its relatively simple genetic composition, its remote phylogenetic position relative to characterized BMCs, and its apparent exclusivity to the enigmatic Verrucomicrobia and Planctomycetes. Members of the phylum Planctomycetes are known for their morphological dissimilarity to the rest of the bacterial domain: internal membranes, reproduction by budding, and lack of peptidoglycan. As a result, they are ripe for many discoveries, but currently the tools for genetic studies are very limited. We expanded the genetic toolbox for the planctomycetes and generated directed gene knockouts of BMC-related genes in Planctomyces limnophilus. A metabolic activity screen revealed that BMC gene products are involved in the degradation of a number of plant and algal cell wall sugars. Among these sugars, we confirmed that BMCs are formed and required for growth on l-fucose and l-rhamnose. Our results shed light on the functional diversity of BMCs as well as their ecological role in the planctomycetes, which are commonly associated with algae.

Genomic evolution of 11 type strains within family Planctomycetaceae.

The species in family Planctomycetaceae are ideal groups for investigating the origin of eukaryotes. Their cells are divided by a lipidic intracytoplasmic membrane and they share a number of eukaryote-like molecular characteristics. However, their genomic structures, potential abilities, and evolutionary status are still unknown. In this study, we searched for common protein families and a core genome/pan genome based on 11 sequenced species in family Planctomycetaceae. Then, we constructed phylogenetic tree based on their 832 common protein families. We also annotated the 11 genomes using the Clusters of Orthologous Groups database. Moreover, we predicted and reconstructed their core/pan metabolic pathways using the KEGG (Kyoto Encyclopedia of Genes and Genomes) orthology system. Subsequently, we identified genomic islands (GIs) and structural variations (SVs) among the five complete genomes and we specifically investigated the integration of two Planctomycetaceae plasmids in all 11 genomes. The results indicate that Planctomycetaceae species share diverse genomic variations and unique genomic characteristics, as well as have huge potential for human applications.

A bacterial tubulovesicular network.

We report the presence of a membranous tubulovesicular network in the planctomycete bacterium Gemmata obscuriglobus. This endomembrane system interacts with membrane coat proteins and is capable of protein internalization and degradation. Taken together, this suggests that the planctomycetal bacterium could illuminate the emergence of complex endomembrane systems.

Three-dimensional reconstruction of bacteria with a complex endomembrane system.

The division of cellular space into functionally distinct membrane-defined compartments has been one of the major transitions in the history of life. Such compartmentalization has been claimed to occur in members of the Planctomycetes, Verrucomicrobiae, and Chlamydiae bacterial superphylum. Here we have investigated the three-dimensional organization of the complex endomembrane system in the planctomycete bacteria Gemmata obscuriglobus. We reveal that the G. obscuriglobus cells are neither compartmentalized nor nucleated as none of the spaces created by the membrane invaginations are closed; instead, they are all interconnected. Thus, the membrane organization of G. obscuriglobus, and most likely all PVC members, is not different from, but an extension of, the "classical" Gram-negative bacterial membrane system. Our results have implications for our definition and understanding of bacterial cell organization, the genesis of complex structure, and the origin of the eukaryotic endomembrane system.

Nested bacterial boxes: nuclear and other intracellular compartments in planctomycetes.

Bacteria in the phylum Planctomycetes and some related phyla challenge our concept of the typical bacterium as consisting of cells without internal compartments or membrane-bounded organelles. Cells of all species of planctomycetes examined consist of at least two major compartments, and there are two other types of compartmentation in which a third compartment is formed either by a double-membrane envelope around the nucleoid in the case of the aerobic Gemmata obscuriglobus or by a single but potentially energized membrane in the case of the anaerobic ammonium-oxidizing anammox planctomycetes. We examine here the nature of these planctomycete compartments in relation to function and their relationship to the endomembranes defining them, and discuss the implications of the remarkable compartment-confined process of protein uptake in Gemmata, which resembles receptor- and clathrin-mediated endocytosis of eukaryotes. Planctomycetes have implications for our understanding of the evolution of membrane-bounded organelles, of endomembranes, transport across endomembranes and membrane trafficking, and for how the complexity of a eukaryote style of cell organization could have originated.

The anammoxosome organelle is crucial for the energy metabolism of anaerobic ammonium oxidizing bacteria.

Anammox bacteria convert ammonium and nitrite to dinitrogen gas under anaerobic conditions to obtain their energy for growth. The anammox reaction was deemed impossible until its discovery in the early 1990s. Now, anammox bacteria are recognized as major players in the global nitrogen cycle and estimated to be responsible for up to 50% of the nitrogen in the air that we breathe. In addition, anammox bacteria are extremely valuable for wastewater treatment where they are applied for the removal of ammonium. Besides their importance in industry and the environment, anammox bacteria defy some basic biological concepts. Whereas most other bacteria have only one cell compartment, the cytoplasm, anammox bacteria have three independent cell compartments bounded by bilayer membranes, from out- to inside; the paryphoplasm, riboplasm and anammoxosome. The anammoxosome is the largest compartment of the anammox cell and is proposed to be dedicated to energy conservation. As such it would be analogous to the mitochondria of eukaryotes. This review will discuss the anammox cell plan in detail, with the main focus on the anammoxosome. The identity of the anammoxosome as a prokaryotic organelle and the importance of this organelle for anammox bacteria are discussed as well as challenges these bacteria face by having three independent cell compartments.