O-Linked β-N-Acetylglucosamine (O-GlcNAc) Regulates Emerin Binding to Barrier to Autointegration Factor (BAF) in a Chromatin- and Lamin B-enriched “Niche”

Emerin, a membrane component of nuclear “lamina” networks with lamins and barrier to autointegration factor (BAF), is highly O-GlcNAc-modified (“O-GlcNAcylated”) in mammalian cells. Mass spectrometry analysis revealed eight sites of O-GlcNAcylation, including Ser-53, Ser-54, Ser-87, Ser-171, and Ser-173. Emerin O-GlcNAcylation was reduced ∼50% by S53A or S54A mutation in vitro and in vivo. O-GlcNAcylation was reduced ∼66% by the triple S52A/S53A/S54A mutant, and S173A reduced O-GlcNAcylation of the S52A/S53A/S54A mutant by ∼30%, in vivo. We separated two populations of emerin, A-type lamins and BAF; one population solubilized easily, and the other required sonication and included histones and B-type lamins. Emerin and BAF associated only in histone- and lamin-B-containing fractions. The S173D mutation specifically and selectively reduced GFP-emerin association with BAF by 58% and also increased GFP-emerin hyper-phosphorylation. We conclude that β-N-acetylglucosaminyltransferase, an essential enzyme, controls two regions in emerin. The first region, defined by residues Ser-53 and Ser-54, flanks the LEM domain. O-GlcNAc modification at Ser-173, in the second region, is proposed to promote emerin association with BAF in the chromatin/lamin B “niche.” These results reveal direct control of a conserved LEM domain nuclear lamina component by β-N-acetylglucosaminyltransferase, a nutrient sensor that regulates cell stress responses, mitosis, and epigenetics.     

O-GlcNAc and the Epigenetic Regulation of Gene Expression

O-GlcNAcylation is an abundant nutrient-driven modification linked to cellular signaling and regulation of gene expression. Utilizing precursors derived from metabolic flux, O-GlcNAc functions as a homeostatic regulator. The enzymes of O-GlcNAc cycling, OGT and O-GlcNAcase, act in mitochondria, the cytoplasm, and the nucleus in association with epigenetic “writers” and “erasers” of the histone code. Both O-GlcNAc and O-phosphate modify repeats within the RNA polymerase II C-terminal domain (CTD). By communicating with the histone and CTD codes, O-GlcNAc cycling provides a link between cellular metabolic status and the epigenetic machinery. Thus, O-GlcNAcylation is poised to influence trans-generational epigenetic inheritance.     

“Candidatus Accumulibacter” Population Structure in Enhanced Biological Phosphorus Removal Sludges as Revealed by Polyphosphate Kinase Genes

We investigated the fine-scale population structure of the “Candidatus Accumulibacter” lineage in enhanced biological phosphorus removal (EBPR) systems using the polyphosphate kinase 1 gene (ppk1) as a genetic marker. We retrieved fragments of “Candidatus Accumulibacter” 16S rRNA and ppk1 genes from one laboratory-scale and several full-scale EBPR systems. Phylogenies reconstructed using 16S rRNA genes and ppk1 were largely congruent, with ppk1 granting higher phylogenetic resolution and clearer tree topology and thus serving as a better genetic marker than 16S rRNA for revealing population structure within the “Candidatus Accumulibacter” lineage. Sequences from at least five clades of “Candidatus Accumulibacter” were recovered by ppk1-targeted PCR, and subsequently, specific primer sets were designed to target the ppk1 gene for each clade. Quantitative real-time PCR (qPCR) assays using “Candidatus Accumulibacter”-specific 16S rRNA and “Candidatus Accumulibacter” clade-specific ppk1 primers were developed and conducted on three laboratory-scale and nine full-scale EBPR samples and two full-scale non-EBPR samples to determine the abundance of the total “Candidatus Accumulibacter” lineage and the relative distributions and abundances of the five “Candidatus Accumulibacter” clades. The qPCR-based estimation of the total “Candidatus Accumulibacter” fraction as a proportion of the bacterial community as measured using 16S rRNA genes was not significantly different from the estimation measured using ppk1, demonstrating the power of ppk1 as a genetic marker for detection of all currently defined “Candidatus Accumulibacter” clades. The relative distributions of “Candidatus Accumulibacter” clades varied among different EBPR systems and also temporally within a system. Our results suggest that the “Candidatus Accumulibacter” lineage is more diverse than previously realized and that different clades within the lineage are ecologically distinct.     

Glycogen metabolism of the anammox bacterium “Candidatus Brocadia sinica”

Presence of glycogen granules in anaerobic ammonium-oxidizing (anammox) bacteria has been reported so far. However, very little is known about their glycogen metabolism and the exact roles. Here, we studied the glycogen metabolism in “Ca. Brocadia sinica” growing in continuous retentostat cultures with bicarbonate as a carbon source. The effect of the culture growth phase was investigated. During the growing phase, intracellular glycogen content increased up to 32.6 mg-glucose (g-biomass dry wt)⁻¹ while the specific growth rate and ATP/ADP ratio decreased. The accumulated glycogen begun to decrease at the onset of entering the near-zero growth phase and was consumed rapidly when substrates were depleted. This clearly indicates that glycogen was synthesized and utilized as an energy storage. The proteomic analysis revealed that “Ca. B. sinica” synthesized glycogen via three known glycogen biosynthesis pathways and simultaneously degraded during the progress of active anammox, implying that glycogen is being continuously recycled. When cells were starved, a part of stored glycogen was converted to trehalose, a potential stress protectant. This suggests that glycogen serves at least as a primary carbon source of trehalose synthesis for survival. This study provides the first physiological evidence of glycogen metabolism in anammox bacteria and its significance in survival under natural substrate-limited habitat.Subject terms: Applied microbiology, Water microbiology     

Ultrastructure of Thiothrix spp. and “Type 021N” Bacteria

The ultrastructural features of two groups of filamentous sulfur bacteria, Thiothrix spp. and an unnamed organism designated “type 021N,” were examined by transmission electron microscopy. Negative staining of whole cells and filaments with uranyl acetate revealed the presence of tufts of fimbriae located at the ends of individual gonidia of Thiothrix sp. strain A1 and “type 021N” strain N7. Holdfast material present at the center of mature rosettes was observed in thin sections stained with ruthenium red. A clearly defined sheath enveloped the trichomes of two of three Thiothrix strains but was absent from “type 021N” filaments. The outer cell wall appeared more complex in “type 021N” strains than in Thiothrix isolates. Bulbs or clusters of irregularly shaped cells, often present in filaments of “type 021N” bacteria, appeared to result from crosswalls which formed at angles oblique to the filament axis. The multicellular nature of these sulfur bacteria was apparent in that only the cytoplasmic membrane and peptidoglycan layer of the cell wall were involved in the septation process. Sulfur inclusions which developed in the presence of sodium thiosulfate were enclosed by a single-layered envelope and located within invaginations of the cytoplasmic membrane.     

An assessment of terminology for intraspecific diversity in fishes,with a focus on “ecotypes” and “life histories”

Understanding and preserving intraspecific diversity (ISD) is important for species conservation. However, ISD units do not have taxonomic standards and are not universally recognized. The terminology used to describe ISD is varied and often used ambiguously. We compared definitions of terms used to describe ISD with use in recent studies of three fish taxa: sticklebacks (Gasterosteidae), Pacific salmon and trout (Oncorhynchus spp., “PST”), and lampreys (Petromyzontiformes). Life history describes the phenotypic responses of organisms to environments and includes biological parameters that affect population growth or decline. Life‐history pathway(s) are the result of different organismal routes of development that can result in different life histories. These terms can be used to describe recognizable life‐history traits. Life history is generally used in organismal‐ and ecology‐based journals. The terms paired species/species pairs have been used to describe two different phenotypes, whereas in some species and situations a continuum of phenotypes may be expressed. Our review revealed overlapping definitions for race and subspecies, and subspecies and ecotypes. Ecotypes are genotypic adaptations to particular environments, and this term is often used in genetic‐ and evolution‐based journals. “Satellite species” is used for situations in which a parasitic lamprey yields two or more derived, nonparasitic lamprey species. Designatable Units, Evolutionary Significant Units (ESUs), and Distinct Population Segments (DPS) are used by some governments to classify ISD of vertebrate species within distinct and evolutionary significant criteria. In situations where the genetic or life‐history components of ISD are not well understood, a conservative approach would be to call them phenotypes.

The terminology used to describe intraspecific diversity is varied and often used ambiguously. “Ecotype” was originally used to describe patterns in genes and ecology, and recent studies employing this term tend to report a genetic basis in ISD. By contrast, “life history” describes biological parameters that affect demography, and this term tends to be used in organismal‐ and ecology‐based journals.     

Isolation, Pure Culture, and Characterization of “Candidatus Arsenophonus arthropodicus,” an Intracellular Secondary Endosymbiont from the Hippoboscid Louse Fly Pseudolynchia canariensis

Members of the genus Arsenophonus comprise a large group of bacterial endosymbionts that are widely distributed in arthropods of medical, veterinary, and agricultural importance. At present, little is known about the role of these bacteria in arthropods, because few representatives have been isolated and cultured in the laboratory. In the current study, we describe the isolation and pure culture of an Arsenophonus endosymbiont from the hippoboscid louse fly Pseudolynchia canariensis. We propose provisional nomenclature for this bacterium in the genus Arsenophonus as “Candidatus Arsenophonus arthropodicus.” Phylogenetic analyses indicate that “Candidatus Arsenophonus arthropodicus” is closely related to the Arsenophonus endosymbionts found in psyllids, whiteflies, aphids, and mealybugs. The pure culture of this endosymbiont offers new opportunities to examine the role of Arsenophonus in insects. To this end, we describe methods for the culture of “Candidatus Arsenophonus arthropodicus” in an insect cell line and the transformation of this bacterium with a broad-host-range plasmid.     

Cloning of the spoT Gene of “Candidatus Phlomobacter fragariae” and Development of a PCR-Restriction Fragment Length Polymorphism Assay for Detection of the Bacterium in Insects

Marginal chlorosis is a new disease of strawberry in which the uncultured phloem-restricted proteobacterium “Candidatus Phlomobacter fragariae” is involved. In order to identify the insect(s) vector(s) of this bacterium, homopteran insects have been captured. Because a PCR test based on the 16S rRNA gene (rDNA) applied to these insects was unable to discriminate between “P. fragariae” and other insect-associated proteobacteria, isolation of “P. fragariae” genes other than 16S rDNA was undertaken. Using comparative randomly amplified polymorphic DNAs, an amplicon was specifically amplified from “P. fragariae”-infected strawberry plants. It encodes part of a “P. fragariae” open reading frame sharing appreciable homology with the spoT gene from other proteobacteria. A spoT-based PCR test combined with restriction fragment length polymorphisms was developed and was able to distinguish “P. fragariae” from other insect bacteria. None of the many leafhoppers and psyllids captured during several years in and around infected strawberry fields was found to carry “P. fragariae.” Interestingly however, the “P. fragariae” spoT sequence could be easily detected in whiteflies proliferating on “P. fragariae”-infected strawberry plants under confined greenhouse conditions but not on control whiteflies, indicating that these insects can become infected with the bacterium.     

“Candidatus Endobugula glebosa,” a Specific Bacterial Symbiont of the Marine Bryozoan Bugula simplex

The bryozoans Bugula neritina and Bugula simplex harbor bacteria in the pallial sinuses of their larvae as seen by electron microscopy. In B. neritina, the bacterial symbiont has been characterized as a gamma-proteobacterium, “Candidatus Endobugula sertula.” “Candidatus E. sertula” has been implicated as the source of the bryostatins, polyketides that provide chemical defense to the host and are also being tested for use in human cancer treatments. In this study, the bacterial symbiont in B. simplex larvae was identified by 16S rRNA-targeted PCR and sequencing as a gamma-proteobacterium closely related to and forming a monophyletic group with “Candidatus E. sertula.” In a fluorescence in situ hybridization, a 16S ribosomal DNA probe specific to the B. simplex symbiont hybridized to long rod-shaped bacteria in the pallial sinus of a B. simplex larva. The taxonomic status “Candidatus Endobugula glebosa” is proposed for the B. simplex larval symbiont. Degenerate polyketide synthase (PKS) primers amplified a gene fragment from B. simplex that closely matched a PKS gene fragment from the bryostatin PKS cluster. PCR surveys show that the symbiont and this PKS gene fragment are consistently and uniquely associated with B. simplex. Bryostatin activity assays and chemical analyses of B. simplex extracts reveal the presence of compounds similar to bryostatins. Taken together, these findings demonstrate a symbiosis in B. simplex that is similar and evolutionarily related to that in B. neritina.     

A New Huanglongbing Species, “Candidatus Liberibacter psyllaurous,” Found To Infect Tomato and Potato, Is Vectored by the Psyllid Bactericera cockerelli (Sulc)

A new huanglongbing (HLB) “Candidatus Liberibacter” species is genetically characterized, and the bacterium is designated “Candidatus Liberibacter psyllaurous.” This bacterium infects the psyllid Bactericera cockerelli and its solanaceous host plants potato and tomato, potentially resulting in “psyllid yellowing.” Host plant-dependent HLB transmission and variation in psyllid infection frequencies are found.     

The Motility Symbiont of the Termite Gut Flagellate Caduceia versatilis Is a Member of the “Synergistes” Group

The flagellate Caduceia versatilis in the gut of the termite Cryptotermes cavifrons reportedly propels itself not by its own flagella but solely by the flagella of ectosymbiotic bacteria. Previous microscopic observations have revealed that the motility symbionts are flagellated rods partially embedded in the host cell surface and that, together with a fusiform type of ectosymbiotic bacteria without flagella, they cover almost the entire surface. To identify these ectosymbionts, we conducted 16S rRNA clone analyses of bacteria physically associated with the Caduceia cells. Two phylotypes were found to predominate in the clone library and were phylogenetically affiliated with the “Synergistes” phylum and the order Bacteroidales in the Bacteroidetes phylum. Probes specifically targeting 16S rRNAs of the respective phylotypes were designed, and fluorescence in situ hybridization (FISH) was performed. As a result, the “Synergistes” phylotype was identified as the motility symbiont; the Bacteroidales phylotype was the fusiform ectobiont. The “Synergistes” phylotype was a member of a cluster comprising exclusively uncultured clones from the guts of various termite species. Interestingly, four other phylotypes in this cluster, including the one sharing 95% sequence identity with the motility symbiont, were identified as nonectosymbiotic, or free-living, gut bacteria by FISH. We thus suggest that the motility ectosymbiont has evolved from a free-living gut bacterium within this termite-specific cluster. Based on these molecular and previous morphological data, we here propose a novel genus and species, “Candidatus Tammella caduceiae,” for this unique motility ectosymbiont of Caducaia versatilis.     

Application of Siderotyping for Characterization of Pseudomonas tolaasii and “Pseudomonas reactans” Isolates Associated with Brown Blotch Disease of Cultivated Mushrooms

Pyoverdine isoelectric focusing analysis and pyoverdine-mediated iron uptake were used as siderotyping methods to analyze a collection of 57 northern and central European isolates of P. tolaasii and “P. reactans.” The bacteria, isolated from cultivated Agaricus bisporus or Pleurotus ostreatus mushroom sporophores presenting brown blotch disease symptoms, were identified according to the white line test (W. C. Wong and T. F. Preece, J. Appl. Bacteriol. 47:401–407, 1979) and their pathogenicity towards A. bisporus and were grouped into siderovars according to the type of pyoverdine they produced. Seventeen P. tolaasii isolates were recognized, which divided into two siderovars, with the first one containing reference strains and isolates of various geographical origins while the second one contained Finnish isolates exclusively. The 40 “P. reactans” isolates divided into eight siderovars. Pyoverdine isoelectric focusing profiles and cross-uptake studies demonstrated an identity for some “P. reactans” isolates, with reference strains belonging to the P. fluorescens biovars II, III, or V. Thus, the easy and rapid methods of siderotyping proved to be reliable by supporting and strengthening previous taxonomical data. Moreover, two potentially novel pyoverdines characterizing one P. tolaasii siderovar and one “P. reactans” siderovar were found.     

Formation of n-Butanol from d-Glucose by Strains of the “Clostridium tetanomorphum” Group

A clostridial strain has been isolated that produced n-butanol, ethanol, butyrate, and acetate as major fermentation products from glucose but no acetone. At a pH of 6.6, n-butanol was formed by this microorganism only during growth. On the basis of its physiological characteristics and DNA-DNA homology data, the strain was assigned to the “Clostridium tetanomorphum” group (S. Nakamura, I. Okado, T. Abe, and S. Nishida, J. Gen. Microbiol. 113:29-35, 1979). All members of this group were shown to produce n-butanol from glucose as the major fermentation product, whereas C. cochlearium produced it in only minor amounts.     

Metabolism of Benzoate, Cyclohex-1-ene Carboxylate, and Cyclohexane Carboxylate by “Syntrophus aciditrophicus” Strain SB in Syntrophic Association with H2-Using Microorganisms

The metabolism of benzoate, cyclohex-1-ene carboxylate, and cyclohexane carboxylate by “Syntrophus aciditrophicus” in cocultures with hydrogen-using microorganisms was studied. Cyclohexane carboxylate, cyclohex-1-ene carboxylate, pimelate, and glutarate (or their coenzyme A [CoA] derivatives) transiently accumulated during growth with benzoate. Identification was based on comparison of retention times and mass spectra of trimethylsilyl derivatives to the retention times and mass spectra of authentic chemical standards. ¹³C nuclear magnetic resonance spectroscopy confirmed that cyclohexane carboxylate and cyclohex-1-ene carboxylate were produced from [ring-¹³C₆]benzoate. None of the metabolites mentioned above was detected in non-substrate-amended or heat-killed controls. Cyclohexane carboxylic acid accumulated to a concentration of 260 μM, accounting for about 18% of the initial benzoate added. This compound was not detected in culture extracts of Rhodopseudomonas palustris grown phototrophically or Thauera aromatica grown under nitrate-reducing conditions. Cocultures of “S. aciditrophicus” and Methanospirillum hungatei readily metabolized cyclohexane carboxylate and cyclohex-1-ene carboxylate at a rate slightly faster than the rate of benzoate metabolism. In addition to cyclohexane carboxylate, pimelate, and glutarate, 2-hydroxycyclohexane carboxylate was detected in trace amounts in cocultures grown with cyclohex-1-ene carboxylate. Cyclohex-1-ene carboxylate, pimelate, and glutarate were detected in cocultures grown with cyclohexane carboxylate at levels similar to those found in benzoate-grown cocultures. Cell extracts of “S. aciditrophicus” grown in a coculture with Desulfovibrio sp. strain G11 with benzoate or in a pure culture with crotonate contained the following enzyme activities: an ATP-dependent benzoyl-CoA ligase, cyclohex-1-ene carboxyl-CoA hydratase, and 2-hydroxycyclohexane carboxyl-CoA dehydrogenase, as well as pimelyl-CoA dehydrogenase, glutaryl-CoA dehydrogenase, and the enzymes required for conversion of crotonyl-CoA to acetate. 2-Ketocyclohexane carboxyl-CoA hydrolase activity was detected in cell extracts of “S. aciditrophicus”-Desulfovibrio sp. strain G11 benzoate-grown cocultures but not in crotonate-grown pure cultures of “S. aciditrophicus”. These results are consistent with the hypothesis that ring reduction during syntrophic benzoate metabolism involves a four- or six-electron reduction step and that once cyclohex-1-ene carboxyl-CoA is made, it is metabolized in a manner similar to that in R. palustris.     

Evolutionary Relationships of “Candidatus Riesia spp.,” Endosymbiotic Enterobacteriaceae Living within Hematophagous Primate Lice

The primary endosymbiotic bacteria from three species of parasitic primate lice were characterized molecularly. We have confirmed the characterization of the primary endosymbiont (P-endosymbiont) of the human head/body louse Pediculus humanus and provide new characterizations of the P-endosymbionts from Pediculus schaeffi from chimpanzees and Pthirus pubis, the pubic louse of humans. The endosymbionts show an average percent sequence divergence of 11 to 15% from the most closely related known bacterium “Candidatus Arsenophonus insecticola.” We propose that two additional species be added to the genus “Candidatus Riesia.” The new species proposed within “Candidatus Riesia” have sequence divergences of 3.4% and 10 to 12% based on uncorrected pairwise differences. Our Bayesian analysis shows that the branching pattern for the primary endosymbionts was the same as that for their louse hosts, suggesting a long coevolutionary history between primate lice and their primary endosymbionts. We used a calibration of 5.6 million years to date the divergence between endosymbionts from human and chimpanzee lice and estimated an evolutionary rate of nucleotide substitution of 0.67% per million years, which is 15 to 30 times faster than previous estimates calculated for Buchnera, the primary endosymbiont in aphids. Given the evidence for cospeciation with primate lice and the evidence for fast evolutionary rates, this lineage of endosymbiotic bacteria can be evaluated as a fast-evolving marker of both louse and primate evolutionary histories.     

Carbon “quantum” dots for bioapplications

Carbon “quantum” dots or carbon dots (CDots) exploit and enhance the intrinsic photoexcited state properties and processes of small carbon nanoparticles via effective nanoparticle surface passivation by chemical functionalization with organic species. The optical properties and photoinduced redox characteristics of CDots are competitive to those of established conventional semiconductor quantum dots and also fullerenes and other carbon nanomaterials. Highlighted here are major advances in the exploration of CDots for their serving as high-performance yet nontoxic fluorescence probes for one- and multi-photon bioimaging in vitro and in vivo, and for their uniquely potent antimicrobial function to inactivate effectively and efficiently some of the toughest bacterial pathogens and viruses under visible/natural or ambient light conditions. Opportunities and challenges in the further development of the CDots platform and related technologies are discussed.     

Discovery of the Novel Candidate Phylum “Poribacteria” in Marine Sponges

Marine sponges (Porifera) harbor large amounts of commensal microbial communities within the sponge mesohyl. We employed 16S rRNA gene library construction using specific PCR primers to provide insights into the phylogenetic identity of an abundant sponge-associated bacterium that is morphologically characterized by the presence of a membrane-bound nucleoid. In this study, we report the presence of a previously unrecognized evolutionary lineage branching deeply in the domain Bacteria that is moderately related to the Planctomycetes, Verrucomicrobia, and Chlamydia lines of decent. Because members of this lineage showed <75% 16S rRNA gene sequence similarity to known bacterial phyla, we suggest the status of a new candidate phylum, named “Poribacteria”, to acknowledge the affiliation of the new bacterium with sponges. The affiliation of the morphologically conspicuous sponge bacterium with the novel phylogenetic lineage was confirmed by fluorescence in situ hybridization with newly designed probes targeting different sites of the poribacterial 16S rRNA. Consistent with electron microscopic observations of cell compartmentalization, the fluorescence signals appeared in a ring-shaped manner. PCR screening with “Poribacteria”-specific primers gave positive results for several other sponge species, while samples taken from the environment (seawater, sediments, and a filter-feeding tunicate) were PCR negative. In addition to a report for Planctomycetes, this is the second report of cell compartmentalization, a feature that was considered exclusive to the eukaryotic domain, in prokaryotes.     

Role of Carbon Dioxide in Catabolism of Propane by “Nocardia paraffinicum” (Rhodococcus rhodochrous)

The catabolism of propane by “Nocardia paraffinicum” (Rhodococcus rhodochrous) has been shown to involve CO₂ fixation after its oxidation to propionic acid. “N. paraffinicum” failed to grow on either propane or 1-propanol in the absence of CO₂. The rate of propane utilization was directly related to the initial CO₂ concentration, and Warburg respirometry suggested that CO₂ was required for the catabolism of 1-propanol, propionaldehyde, and propionate but not for 2-propanol. These data also suggested that the predominant pathway for the utilization of propane by “N. paraffinicum” was through 1-propanol. The use of [2-¹⁴C]propane and ¹⁴CO₂ confirmed the catabolism of propane and the fixation of CO₂. Through the use of these isotopes and the pyruvate carboxylase inhibitor sodium arsenite, the labeled 2,4-dinitrophenylhydrazine derivative of pyruvate was trapped and isolated via thin-layer chromatography. The trapping of [¹⁴C]pyruvate in this manner was considered to be indicative of the presence of the methylmalonyl coenzyme A pathway for CO₂ fixation.