Biological Role of Pigment Production for the Bacterial Phytopathogen Pantoea stewartii subsp. stewartii

Pantoea stewartii subsp. stewartii, the causal agent of Stewart's wilt of sweet corn, produces a yellow carotenoid pigment. A nonpigmented mutant was selected from a bank of mutants generated by random transposon mutagenesis. The transposon insertion site was mapped to the crtB gene, encoding a putative phytoene synthase, an enzyme involved in the early steps of carotenoid biosynthesis. We demonstrate here that the carotenoid pigment imparts protection against UV radiation and also contributes to the complete antioxidant pathway of P. stewartii. Moreover, production of this pigment is regulated by the EsaI/EsaR quorum-sensing system and significantly contributes to the virulence of the pathogen in planta.     

Pantoea stewartii subsp. stewartii produces an endoglucanase that is required for full virulence in sweet corn

Pantoea stewartii subsp. stewartii, a xylem-dwelling bacterium, is the causal agent of Stewart's wilt and blight of sweet corn. The goal of this study was to characterize the only gene in the P. stewartii subsp. stewartii genome predicted to encode an endoglucanase (EGase); this gene was designated engY. Culture supernatants from P. stewartii subsp. stewartii and Escherichia coli expressing recombinant EngY protein possessed both EGase and xylanase activities. Deletion of engY abolished EGase and xylanase activity, demonstrating that EngY appears to be the major EGase or xylanase produced by P. stewartii subsp. stewartii. Most importantly, our results show that EngY contributes to movement in the xylem and disease severity during the wilting phase of Stewart's wilt but is not required for water-soaked lesion formation.     

The kinesin Eg5 drives poleward microtubule flux in Xenopus laevis egg extract spindles

Although mitotic and meiotic spindles maintain a steady-state length during metaphase, their antiparallel microtubules slide toward spindle poles at a constant rate. This "poleward flux" of microtubules occurs in many organisms and may provide part of the force for chromosome segregation. We use quantitative image analysis to examine the role of the kinesin Eg5 in poleward flux in metaphase Xenopus laevis egg extract spindles. Pharmacological inhibition of Eg5 results in a dose-responsive slowing of flux, and biochemical depletion of Eg5 significantly decreases the flux rate. Our results suggest that ensembles of nonprocessive Eg5 motors drive flux in metaphase Xenopus extract spindles.     

Precipitation of Calcite by Indigenous Microorganisms to Strengthen Liquefiable Soils

Enrichments for indigenous microorganisms capable of hydrolyzing urea in the presence of CaCl₂ were performed on potentially liquefiable saturated soils in both the laboratory and in situ. Following enrichment, treatment of soils with nutrients, CaCl₂ and urea resulted in significant in situ precipitation of calcite, even at depth, by indigenous microorganisms. The biomineralized soils showed properties that indicate calcite precipitation increased their resistance to seismic-induced liquefaction.     

Semiconservative synthesis of single-stranded RNA by bacteriophage phi 6 RNA polymerase.

The RNA polymerase in the nucleocapsid of Pseudomonas phaseolicola bacteriophage phi 6 transcribed large, medium, and small single-stranded RNA from the viral double-stranded RNA genome by a semiconservative (displacement) mechanism. Approximately 23%, 63%, and 65% of the nucleocapsid particles in the assay mixture synthesized at least one round of large, medium, and small single-stranded RNA molecules, respectively. Some of these particles reinitiated synthesis such that an average of 1.5 large, 33 medium, and 24 small single-stranded RNAs were synthesized from each double-stranded RNA.     

Protection of Bacillus pumilus Spores by Catalases

Bacillus pumilus SAFR-032, isolated at spacecraft assembly facilities of the National Aeronautics and Space Administration Jet Propulsion Laboratory, is difficult to kill by the sterilization method of choice, which uses liquid or vapor hydrogen peroxide. We identified two manganese catalases, YjqC and BPUM_1305, in spore protein extracts of several B. pumilus strains by using PAGE and mass spectrometric analyses. While the BPUM_1305 catalase was present in six of the B. pumilus strains tested, YjqC was not detected in ATCC 7061 and BG-B79. Furthermore, both catalases were localized in the spore coat layer along with laccase and superoxide dismutase. Although the initial catalase activity in ATCC 7061 spores was higher, it was less stable over time than the SAFR-032 enzyme. We propose that synergistic activity of YjqC and BPUM_1305, along with other coat oxidoreductases, contributes to the enhanced resistance of B. pumilus spores to hydrogen peroxide. We observed that the product of the catalase reaction, gaseous oxygen, forms expanding vesicles on the spore surface, affecting the mechanical integrity of the coat layer, resulting in aggregation of the spores. The accumulation of oxygen gas and aggregations may play a crucial role in limiting further exposure of Bacilli spore surfaces to hydrogen peroxide or other toxic chemicals when water is present.     

Depression-biased reverse plasticity rule is required for stable learning at top-down connections

Top-down synapses are ubiquitous throughout neocortex and play a central role in cognition, yet little is known about their development and specificity. During sensory experience, lower neocortical areas are activated before higher ones, causing top-down synapses to experience a preponderance of post-synaptic activity preceding pre-synaptic activity. This timing pattern is the opposite of that experienced by bottom-up synapses, which suggests that different versions of spike-timing dependent synaptic plasticity (STDP) rules may be required at top-down synapses. We consider a two-layer neural network model and investigate which STDP rules can lead to a distribution of top-down synaptic weights that is stable, diverse and avoids strong loops. We introduce a temporally reversed rule (rSTDP) where top-down synapses are potentiated if post-synaptic activity precedes pre-synaptic activity. Combining analytical work and integrate-and-fire simulations, we show that only depression-biased rSTDP (and not classical STDP) produces stable and diverse top-down weights. The conclusions did not change upon addition of homeostatic mechanisms, multiplicative STDP rules or weak external input to the top neurons. Our prediction for rSTDP at top-down synapses, which are distally located, is supported by recent neurophysiological evidence showing the existence of temporally reversed STDP in synapses that are distal to the post-synaptic cell body.     

Topoisomerase II from Chlorella virus PBCV-1. Characterization of the smallest known type II topoisomerase

Type II topoisomerases, a family of enzymes that govern topological DNA interconversions, are essential to many cellular processes in eukaryotic organisms. Because no data are available about the functions of these enzymes in the replication of viruses that infect eukaryotic hosts, this led us to express and characterize the first topoisomerase II encoded by one of such viruses. Paramecium bursaria chlorella virus 1 (PBCV-1) infects certain chlorella-like green algae and encodes a 120-kDa protein with a similarity to type II topoisomerases. This protein was expressed in Saccharomyces cerevisiae and was highly active in relaxation of both negatively and positively supercoiled plasmid DNA, catenation of plasmid DNA, and decatenation of kinetoplast DNA networks. Its optimal activity was determined, and the omission of Mg(2+) or its replacement with other divalent cations abolished DNA relaxation. All activities of the recombinant enzyme were ATP dependent. Increasing salt concentrations shifted DNA relaxation from a normally processive mechanism to a distributive mode. Thus, even though the PBCV-1 enzyme is considerably smaller than other eukaryotic topoisomerase II enzymes (whose molecular masses are typically 160-180 kDa), it displays all the catalytic properties expected for a type II topoisomerase.     

Slide-and-cluster models for spindle assembly

BACKGROUND: Mitotic and meiotic spindles are assemblies of microtubules (MTs) that form during cell division to physically separate sister chromosomes. How the various components of spindles act together to establish and maintain the dynamic bipolar structure of spindles is not understood. Interactions between MTs and motors have been studied both experimentally and theoretically in many contexts, including the self-organization of arrays of MTs by motors and the competition between different classes of motors to move a single load. This work demonstrates how the interplay between two types of motors together with continual nucleation of MTs by chromosomes could organize the MTs into spindles. RESULTS: We propose a slide-and-cluster model based on four known molecular activities: MT nucleation near chromosomes, the sliding of MTs by a plus-end-directed motor, the clustering of their minus ends by a minus-end-directed motor, and the loss of MTs by dynamic instability. Our model applies to overlapping, nonkinetochore MTs in anastral spindles, and perhaps also to interpolar MTs in astral spindles. We show mathematically that the slide-and-cluster mechanism robustly forms bipolar spindles with sharp poles and a stable steady-state length. This model accounts for several experimental observations that were difficult to explain with existing models. Three new predictions of the model were tested and verified in Xenopus egg extracts. CONCLUSIONS: We show that a simple two-motor model could create stable, bipolar spindles under a wide range of physical parameters. Our model is the first self-contained model for anastral spindle assembly and MT sliding (known as poleward flux). Our experimental results support the slide-and-cluster scenario; most significantly, we find that MT sliding slows near spindle poles, confirming the model's primary prediction.