Role of type IV pili in virulence of Pseudomonas syringae pv. tabaci 6605: correlation of motility, multidrug resistance, and HR-inducing activity on a nonhost plant

To investigate the role of type IV pili in the virulence of phytopathogenic bacteria, four mutant strains for pilus biogenesis-related genes were generated in Pseudomonas syringae pv. tabaci 6605. PilA encodes the pilin protein as a major subunit of type IV pili, and the pilO product is reported to be required for pilus assembly. The fimU and fimT genes are predicted to produce minor pilins. Western blot analysis revealed that pilA, pilO, and fimU mutants but not the fimT mutant failed to construct type IV pili. Although the swimming motility of all mutant strains was not impaired in liquid medium, they showed remarkably reduced motilities on semisolid agar medium, suggesting that type IV pili are required for surface motilities. Virulence toward host tobacco plants and hypersensitive response-inducing ability in nonhost Arabidopsis leaves of pilA, pilO, and fimU mutant strains were reduced. These results might be a consequence of reduced expression of type III secretion system-related genes in the mutant strains. Further, all mutant strains showed enhanced expression of resistance-nodulation-division family members mexA, mexB, and oprM, and higher tolerance to antimicrobial compounds. These results indicate that type IV pili are an important virulence factor of this pathogen.     

Hedgehog-Gli Activators Direct Osteo-chondrogenic Function of Bone Morphogenetic Protein toward Osteogenesis in the Perichondrium

Specification of progenitors into the osteoblast lineage is an essential event for skeletogenesis. During endochondral ossification, cells in the perichondrium give rise to osteoblast precursors. Hedgehog (Hh) and bone morphogenetic protein (BMP) are suggested to regulate the commitment of these cells. However, properties of perichondrial cells and regulatory mechanisms of the specification process are still poorly understood. Here, we investigated the machineries by combining a novel organ culture system and single-cell expression analysis with mouse genetics and biochemical analyses. In a metatarsal organ culture reproducing bone collar formation, activation of BMP signaling enhanced the bone collar formation cooperatively with Hh input, whereas the signaling induced ectopic chondrocyte formation in the perichondrium without Hh input. Similar phenotypes were also observed in compound mutant mice, where signaling activities of Hh and BMP were genetically manipulated. Single-cell quantitative RT-PCR analyses showed heterogeneity of perichondrial cells in terms of natural characteristics and responsiveness to Hh input. In vitro analyses revealed that Hh signaling suppressed BMP-induced chondrogenic differentiation; Gli1 inhibited the expression of Sox5, Sox6, and Sox9 (SRY box-containing gene 9) as well as transactivation by Sox9. Indeed, ectopic expression of chondrocyte maker genes were observed in the perichondrium of metatarsals in Gli1^−/− fetuses, and the phenotype was more severe in Gli1^−/−;Gli2^−/− newborns. These data suggest that Hh-Gli activators alter the function of BMP to specify perichondrial cells into osteoblasts; the timing of Hh input and its target populations are critical for BMP function.     

Salt dynamics in Tamarix ramosissima in the lower Virgin River floodplain, Nevada

Tamarisk (Tamarix spp.) is a halophyte with salt glands on its leaves and is an invasive riparian plant in the US. To increase our understanding of the effects of Tamarix on soil salinity, we conducted a year-long field investigation to evaluate the salt dynamics of a stand of Tamarix ramosissima along the lower Virgin River floodplain, NV, USA. We examined salt accumulation in the biomass and studied salt return to the soil by litter fall, throughfall and stemflow from September 2009 to September 2010. We also investigated soil salinity concentrations inside and outside of the stand where native shrub species was sparsely distributed. The average Na⁺ accumulated in the plant biomass was estimated at 23.4 g m^?2. The Na⁺ returned to the soil through litter fall, throughfall and stemflow during the investigation was similar with that accumulated in the plant biomass. More than 90 % of Na⁺ leached to the soil was from throughfall and stemflow. Soil salinity was significantly lower inside than outside of the stand. Salt secretion from Tamarix is generally expected to increase soil salinity in stands. However, our results suggest that surface soil salinity does not necessarily increase in the Tamarix stand along the lower Virgin River floodplain that is subjected to occasional flooding.     

4-O-Methylglucuronoxylan Isolated from the Midrib of Nicotiana tabacum

An acidic xylan from the midrib of Nicotiana tabacum was isolated by alkaline extraction and fractionation on a DEAE-cellulose column. Based on the results of methylation analysis, partial acid hydrolysis and Smith degradation, the acidic xylan was concluded to be composed of a linear backbone of β-(1→4)-linked D-xylopyranosyl residues with approximately every ninth residue carrying a terminal 4-O-methyl-α-D-glucopyranosyluronic acid residue linked as a single side chain by (1→2) linkage.     

Overexpression,purification, and characterization of Paenibacillus cell surface-expressed chitinase ChiW with two catalytic domains

Paenibacillus sp. strain FPU-7 produces several different chitinases and effectively hydrolyzes robust chitin. Among the P. FPU-7 chitinases, ChiW, a novel monomeric chitinase with a molecular mass of 150?kDa, is expressed as a cell surface molecule. Here, we report that active ChiW lacking the anchoring domains in the N-terminus was successfully overproduced in Escherichia coli and purified to homogeneity. The two catalytic domains at the C-terminal region were classified as typical glycoside hydrolase family 18 chitinases, whereas the N-terminal region showed no sequence similarity to other known proteins. The vacuum-ultraviolet circular dichroism spectrum of the enzyme strongly suggested the presence of a β-stranded-rich structure in the N-terminus. Its biochemical properties were also characterized. Various insoluble chitins were hydrolyzed to N,N’-diacetyl-D-chitobiose as the final product. Based on amino acid sequence similarities and site-directed mutagenesis, Glu691 and Glu1177 in the two GH-18 domains were identified as catalytic residues.     

A New Antibiotic K-52B

A new antibiotic K-52B, different from K-52A, was isolated from the culture broth of Streptoverticillium roseoverticillatum subsp. albosporum, strain No. K-52. The antibiotic K-52B was thought to be a similar saccharide to K-52A from its physicochemical properties but differed from K-52A in the presence of nitrogen content. Antibiotic K-52B inhibited the growth of Gram-positive and Gram-negative bacteria, including Pseudomonas aeruginosa on a chemically defined medium. The growth inhibition was, however, reversed by l-glutamine, l-glutamic acid, l-asparagine and l-aspartic acid.     

Anti-prion activity of an RNA aptamer and its structural basis

Prion proteins (PrPs) cause prion diseases, such as bovine spongiform encephalopathy. The conversion of a normal cellular form (PrP^C) of PrP into an abnormal form (PrP^Sc) is thought to be associated with the pathogenesis. An RNA aptamer that tightly binds to and stabilizes PrP^C is expected to block this conversion and to thereby prevent prion diseases. Here, we show that an RNA aptamer comprising only 12 residues, r(GGAGGAGGAGGA) (R12), reduces the PrP^Sc level in mouse neuronal cells persistently infected with the transmissible spongiform encephalopathy agent. Nuclear magnetic resonance analysis revealed that R12, folded into a unique quadruplex structure, forms a dimer and that each monomer simultaneously binds to two portions of the N-terminal half of PrP^C, resulting in tight binding. Electrostatic and stacking interactions contribute to the affinity of each portion. Our results demonstrate the therapeutic potential of an RNA aptamer as to prion diseases.     

Morphological and electrophysiological differences in tarsal chemosensilla between the wild silkmoth Bombyx mandarina and the domesticated species Bombyx mori

Gustatory and olfactory senses of phytophagous insects play important roles in the recognition of host plants. In the domestic silkmoth Bombyx mori and its wild species Bombyx mandarina, the morphologies and responses of adult olfactory organs (antennae) have been intensely investigated. However, little is known about these features of adult gustatory organs and the influence of domestication on the gustatory sense. Here we revealed that both species have two types of sensilla (thick [T] and slim [S] types) on the fifth tarsomeres of the adult legs. In both species, females have 3.6–6.9 times more T-sensilla than males. Therefore, T-sensilla seem to play more important roles in females than in males. Moreover, gustatory cells of T-sensilla of B. mandarina females responded intensely to mulberry leaf extract in electrophysiological experiments, while T-sensilla of B. mori females (N4 strain) hardly responded to mulberry leaf extract. These results suggest that T-sensilla of B. mandarina females are involved in the recognition of oviposition sites. We also observed that, in three B. mori strains (N4, p50T, and Kinshu × Showa), the densities of sensilla on the fifth tarsomeres were much lower than in B. mandarina. These results indicate that domestication has influenced the tarsal gustatory system of B. mori.