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.     

Hybridization of Rous Sarcoma Virus Deoxyribonucleic Acid Polymerase Product and Ribonucleic Acids from Chicken and Rat Cells Infected with Rous Sarcoma Virus

Rous sarcoma virus (RSV)-specific ribonucleic acid (RNA) in virus-producing chicken cells and non-virus-producing rat cells infected with RSV was studied by hybridization with the endogenous deoxyribonucleic acid (DNA) product of the RSV virion DNA polymerase system. By hybridizing the total DNA product with excess virion RNA, the product DNA was separated into hybridized (“minus”) and nonhybridized (“plus”) DNA. The “minus” DNA was complementary to at least 20% of the RNA from RSV which remained of high molecular weight after denaturation. A maximum of approximately 65% hybridization was observed between “minus” DNA and RSV RNA or RSV-infected chicken cell RNA. A maximum of about 60% hybridization was observed between “minus” DNA and RSV-infected rat cell RNA. RSV-infected chicken cells contained RSV-specific RNA equivalent to about 6,000 virions per cell. RSV-infected rat cells contained RSV-specific RNA equivalent to approximately 400 virions per cell. Neither cell type contained detectable RNA complementary to virion RNA. The RSV-specific RNA in RSV-infected rat cells did not appear to be qualitatively different from that in RSV-infected chicken cells.     

“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.     

α-Proteobacterial Symbionts of Marine Bryozoans in the Genus Watersipora

Bacterial symbionts that resembled mollicutes were discovered in the marine bryozoan Watersipora arcuata in the 1980s. In this study, we used PCR and sequencing of 16S rRNA genes, specific fluorescence in situ hybridization, and phylogenetic analysis to determine that the bacterial symbionts of “W. subtorquata” and “W. arcuata” from several locations along the California coast are actually closely related α-Proteobacteria, not mollicutes. We propose the names “Candidatus Endowatersipora palomitas” and “Candidatus Endowatersipora rubus” for the symbionts of “W. subtorquata” and “W. arcuata,” respectively.     

Molecular Evidence for the Broad Distribution of Anaerobic Ammonium-Oxidizing Bacteria in Freshwater and Marine Sediments

Previously available primer sets for detecting anaerobic ammonium-oxidizing (anammox) bacteria are inefficient, resulting in a very limited database of such sequences, which limits knowledge of their ecology. To overcome this limitation, we designed a new primer set that was 100% specific in the recovery of ~700-bp 16S rRNA gene sequences with >96% homology to the “Candidatus Scalindua” group of anammox bacteria, and we detected this group at all sites studied, including a variety of freshwater and marine sediments and permafrost soil. A second primer set was designed that exhibited greater efficiency than previous primers in recovering full-length (1,380-bp) sequences related to “Ca. Scalindua,” “Candidatus Brocadia,” and “Candidatus Kuenenia.” This study provides evidence for the widespread distribution of anammox bacteria in that it detected closely related anammox 16S rRNA gene sequences in 11 geographically and biogeochemically diverse freshwater and marine sediments.     

Characterization of the Rapidly Labeled Hybridizable RNA Synthesized in L5178Y Mouse Leukemic Cells: Hybridization Lifetime Studies

An attempt is made to characterize the rapidly labeled hybridizable RNA of L5178Y mouse leukemic cells which has been shown to have similar base sequences when synthesized in two different stages of the cell cycle. The size of rapidly labeled RNA molecules was heterogeneous. For labeling times of 20 min or less, the per cent of hybridization was maximal. With longer labeling times, the per cent of hybridization decreased as radioactivity appeared in long-lived species of low hybridization efficiency; the radioactivity profile resembled the optical density profile in sucrose gradients. The lifetime of newly synthesized hybridizable RNA was studied by pulse labeling exponentially growing cells and then “chasing” with nonradioactive uridine. The per cent of hybridization was studied as a function of chase time. Three RNA groups, which comprised different proportions of rapidly labeled hybridizable RNA, were distinguished. The short-lived group had a half-life of 10 min, much less than the values reported in the literature for messenger RNA of mammalian cells. The half-life of 1-1½ hr observed for a medium-lived group more closely corresponds to that of messenger RNA. A long-lived group had a half-life of approximately 20 hr. Specific activity measurements during chase indicate the presence of a “pool” of labeled uridine derivatives. The uridine of this pool appears to be nonexchangeable with but dilutable by exogenous uridine. A nontoxic concentration of actinomycin D was added to the chase media in an attempt to block the “pool effect”. A rapidly degradable RNA was demonstrable both by specific activity and per cent of hybridization measurements.     

Characterization of Chlorinated Aliphatic Hydrocarbons and Environmental Variables in a Shallow Groundwater in Shanghai Using Kriging Interpolation and Multifactorial Analysis

CAHs, as a cleaning solvent, widely contaminated shallow groundwater with the development of manufacturing in China''s Yangtze River Delta. This study focused on the distribution of CAHs, and correlations between CAHs and environmental variables in a shallow groundwater in Shanghai, using kriging interpolation and multifactorial analysis. The results showed that the overall CAHs plume area (above DIV) was approximately 9,000 m² and located in the 2–4 m underground, DNAPL was accumulated at an area of approximately 1,400 m² and located in the 6-8m sandy silt layer on the top of the muddy silty clay. Heatmap of PPC for CAHs and environmental variables showed that the correlation between “Fe²⁺” and most CAHs such as “1,1,1-TCA”, “1,1-DCA”, “1,1-DCE” and “%TCA” were significantly positive (p<0.001), but “%CA” and/or “%VC” was not, and “Cl^-” was significantly positive correlated with “1,1-DCA” and “1,1-DCE” (p<0.001). The PCA demonstrated that the relative proportions of CAHs in groundwater were mostly controlled by the sources and the natural attenuation. In conclusion, the combination of geographical and chemometrics was helpful to establishing an aerial perspective of CAHs and identifying reasons for the accumulation of toxic dechlorination intermediates, and could become a useful tool for characterizing contaminated sites in general.     

Broad Distribution of Diverse Anaerobic Ammonium-Oxidizing Bacteria in Chinese Agricultural Soils

Anaerobic ammonium-oxidizing (anammox) bacteria have been detected in many marine and freshwater ecosystems. However, little is known about the distribution, diversity, and abundance of anammox bacteria in terrestrial ecosystems. In this study, anammox bacteria were found to be present in various agricultural soils collected from 32 different locations in China. Phylogenetic analysis of the 16S rRNA genes showed “Candidatus Brocadia,” “Candidatus Kuenenia,” “Candidatus Anammoxoglobus,” and “Candidatus Jettenia” in the collected soils, with “Candidatus Brocadia” being the dominant genus. Quantitative PCR showed that the abundance of anammox bacteria ranged from 6.38 × 10⁴ ± 0.42 × 10⁴ to 3.69 × 10⁶ ± 0.25 × 10⁶ copies per gram of dry weight. Different levels of diversity, composition, and abundance of the anammox bacterial communities were observed, and redundancy analysis indicated that the soil organic content and the distribution of anammox communities were correlated in the soils examined. Furthermore, Pearson correlation analysis showed that the diversity of the anammox bacteria was positively correlated with the soil ammonium content and the organic content, while the anammox bacterial abundance was positively correlated with the soil ammonium content. These results demonstrate the broad distribution of diverse anammox bacteria and its correlation with the soil environmental conditions within an extensive range of Chinese agricultural soils.     

Measurement of the Size Distribution of Zymogen Granules from Rat Pancreas

Zymogen granules are obtained in pure form and processed for electron microscopy. Thin sections are photographed and diameters measured with a Zeiss particle size analyzer. Since sectioning cuts any given particle in random way, these diameters are not the true diameters of the particles. The true size distribution is obtained by comparing the observed diameter distribution with a generated diameter distribution. The generated distribution is constructed from an assumed parent distribution (of true diameters) by the Monte-Carlo technique. “Goodness of fit” is judged by the value of “chi-squared” resulting from the comparison. Appropriate adjustments of the parameters of the true distribution are made on the basis of minimizing chi-square. A result of this process is that the zymogen granules follow a normal distribution: mean = 0.984 ±0.005 μm, SD = 0.190 ±0.005 μm. A second preparation of granules was made and diameters were measured directly with a scanning electron microscope. The distribution was again found to be normal, thus supporting the first result.     

16S rRNA Phylogenetic Investigation of the Candidate Division “Korarchaeota”

The environmental distribution and phylogeny of “Korarchaeota,” a proposed ancient archaeal division, was investigated by using the 16S rRNA gene framework. Korarchaeota-specific primers were designed based on previously published sequences and used to screen a variety of environments. Korarchaeota 16S rRNA genes were amplified exclusively from high temperature Yellowstone National Park hot springs and a 9°N East Pacific Rise deep-sea hydrothermal vent. Phylogenetic analyses of these and all available sequences suggest that Korarchaeota exhibit a high level of endemicity.     

X-Ray Solution Scattering of Squid Heavy Meromyosin: Strengthening the Evidence for an Ancient Compact off State

The overall conformations of regulated myosins or heavy meromyosins from chicken/turkey, scallop, tarantula, limulus, and scorpion sources have been studied by a number of techniques, including electron microscopy, sedimentation, and pulsed electron paramagnetic resonance. These studies have indicated that the binding of regulatory ions changes the conformation of the molecule from a compact shape found in the “off” state of the muscle to extended relationships between the tail and independently mobile heads that predominate in the “on” state. Here we strengthen the argument for the generality of this conformational change by using small angle X-ray scattering on heavy meromyosin from squid. Small angle X-ray scattering allows the protein to be visualized in solution under mild and relatively physiological conditions, and squid differs from the other species studied by at least 500 million years of evolution. Analysis of the data indicates that upon addition of Ca²⁺ the radius of gyration increases. Differences in the squid “on” and “off” states are clearly distinguishable as bimodal and unimodal pair distance distribution functions respectively. These observations are consistent with a Ca²⁺-free squid heavy meromyosin that is compact, but which becomes extended when Ca²⁺ is bound. Further, the scattering profile derived from the current model of tarantula heavy meromyosin in the “off” state is in excellent agreement with the measured “off” state scattering profile for squid heavy meromyosin. The previous and current studies together provide significant evidence that regulated myosin''s compact off-state conformation is an ancient trait, inherited from a common ancestor during divergent evolution.     

Exercise‐induced α‐ketoglutaric acid stimulates muscle hypertrophy and fat loss through OXGR1‐dependent adrenal activation

The authors approached the journal to correct a mistake in the data presented in Appendix␣Fig S3D. The authors state that the mouse images in Appendix␣Fig S3D mistakenly displayed images from Fig 2F and Appendix␣Fig S1F. The images in Appendix␣Fig S3D are herewith corrected. The authors state that this change does not affect the conclusions or the statistics. The source data for these panels have been added to the original publication.The authors note that the following sentence needs to be corrected from: Appendix Figure S3D. Original. Appendix Figure S3D. Corrected. “Interestingly, several well‐established accumulation signatures of succinate, malate, hypoxanthine, and xanthine induced by endurance exercise (Lewis et␣al, 2010) were found to be decreased by endurance exercise (Figs 1D and EV1A–D)”.to“Interestingly, several well‐established accumulation signatures of succinate, malate, hypoxanthine, and xanthine induced by endurance exercise (Lewis et␣al, 2010) were found to be decreased by resistance exercise (Figs 1D and EV1A–D)”.Further, the authors requested to amend the legend of Appendix␣Fig S3R to indicate that the same sample for the iWAT group, “WT+2%AKG” treatment, is shown in Fig 3P. The corrected legend reads: “(R‐S). Representative images (R) and quantification (S) of p‐HSL DAB staining from male OXGR1OE^AG mice treated with AKG for 12 weeks (n = 6 per group). The same sample is shown as in Fig 3P ”.The authors regret these errors and any confusion they may have caused. All authors approve of this correction.     

Prey Capture and Phagocytosis in the Choanoflagellate Salpingoeca rosetta

Choanoflagellates are unicellular and colonial aquatic microeukaryotes that capture bacteria using an apical flagellum surrounded by a feeding collar composed of actin-filled microvilli. Flow produced by the apical flagellum drives prey bacteria to the feeding collar for phagocytosis. We report here on the cell biology of prey capture in rosette-shaped colonies and unicellular “thecate” or substrate attached cells from the choanoflagellate S. rosetta. In thecate cells and rosette colonies, phagocytosis initially involves fusion of multiple microvilli, followed by remodeling of the collar membrane to engulf the prey, and transport of engulfed bacteria into the cell. Although both thecate cells and rosette colony cells produce ∼70 nm “collar links” that connect and potentially stabilize adjacent microvilli, only thecate cells were observed to produce a lamellipod-like “collar skirt” that encircles the base of the collar. This study offers insight into the process of prey ingestion by S. rosetta, and provides a context within which to consider potential ecological differences between solitary cells and colonies in choanoflagellates.     

Rank Diversity of Languages: Generic Behavior in Computational Linguistics

Statistical studies of languages have focused on the rank-frequency distribution of words. Instead, we introduce here a measure of how word ranks change in time and call this distribution rank diversity. We calculate this diversity for books published in six European languages since 1800, and find that it follows a universal lognormal distribution. Based on the mean and standard deviation associated with the lognormal distribution, we define three different word regimes of languages: “heads” consist of words which almost do not change their rank in time, “bodies” are words of general use, while “tails” are comprised by context-specific words and vary their rank considerably in time. The heads and bodies reflect the size of language cores identified by linguists for basic communication. We propose a Gaussian random walk model which reproduces the rank variation of words in time and thus the diversity. Rank diversity of words can be understood as the result of random variations in rank, where the size of the variation depends on the rank itself. We find that the core size is similar for all languages studied.     

Transcription of Simian Virus 40 II. Hybridization of RNA Extracted from Different Lines of Transformed Cells to the Separated Strands of Simian Virus 40 DNA

The amount of simian virus 40 (SV40) DNA present in various SV40-transformed mouse cell lines and “revertants” isolated from them was determined. The number of viral DNA copies in the different cell lines ranged from 1.35 to 8.75 copies per diploid quantity of mouse cell DNA and from 2.2 to 14 copies per cell. The revertants had the same number of viral DNA copies per diploid quantity of mouse cell DNA as their parental cell lines. (However, they showed an increased number of viral DNA copies per cell due to their increased amount of DNA.) By using separated strands of SV40 DNA, the extent of each DNA strand transcribed into stable RNA species was determined for the transformed and “revertant” cell lines. From 30 to 80% of the “early” strand and from 0 to 20% of the “late” strand was present as stable RNA species in the cell lines tested. There was no alteration in the pattern of the stable viral RNA species present in three concanavalin A-selected revertants, whereas in a fluorodeoxyuridine-selected revertant there appeared to be less viral-specific RNA present in the cells.     

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.     

Anatomic Characteristics Associated with Head Splitting in Cabbage (Brassica oleracea var. capitata L.)

Cabbage belonging to Brassicaceae family is one of the most important vegetables cultivated worldwide. The economically important part of cabbage crop is head, formed by leaves which may be of splitting and non-splitting types. Cabbage varieties showing head splitting causes huge loss to the farmers and therefore finding the molecular and structural basis of splitting types would be helpful to breeders. To determine which anatomical characteristics were related to head-splitting in cabbage, we analyzed two contrasting cabbage lines and their offspring using a field emission scanning electron microscope. The inbred line “747” is an early head-splitting type, while the inbred line “748” is a head-splitting-resistant type. The petiole cells of “747” seems to be larger than those of “748” at maturity; however, there was no significant difference in petiole cell size at both pre-heading and maturity stages. The lower epidermis cells of “747” were larger than those of “748” at the pre-heading and maturity stages. “747” had thinner epidermis cell wall than “748” at maturity stage, however, there was no difference of the epidermis cell wall thickness in the two lines at the pre-heading stage. The head-splitting plants in the F₁ and F₂ population inherited the larger cell size and thinner cell walls of epidermis cells in the petiole. In the petiole cell walls of “747” and the F₁ and F₂ plants that formed splitting heads, the cellulose microfibrils were loose and had separated from each other. These findings verified that anomalous cellulose microfibrils, larger cell size and thinner-walled epidermis cells are important genetic factors that make cabbage heads prone to splitting.