Assembly properties of bacterial actin MreB involved in Spiroplasma swimming motility.

Bacterial actin MreB forms filaments composed of antiparallel double-stranded units. The wall-less helical bacterium Spiroplasma has five MreB homologs (MreB1-5), some of which are involved in an intracellular ribbon for driving the bacterium's swimming motility. Although the interaction between MreB units is important for understanding Spiroplasma swimming, the interaction modes of each ribbon component are unclear. Here, we examined the assembly properties of Spiroplasma eriocheiris MreB5 (SpeMreB5), one of the ribbon component proteins that forms sheets. Electron microscopy revealed that sheet formation was inhibited under acidic conditions and bundle structures were formed under acidic and neutral conditions with low ionic strength. We also used solution assays and identified four properties of SpeMreB5 bundles as follows: (I) bundle formation followed sheet formation; (II) electrostatic interactions were required for bundle formation; (III) the positively charged and unstructured C-terminal region contributed to promoting lateral interactions for bundle formation; and (IV) bundle formation required Mg2+ at neutral pH but was inhibited by divalent cations under acidic pH conditions. During these studies, we also characterized two aggregation modes of SpeMreB5 with distinct responses to ATP. These properties will shed light on SpeMreB5 assembly dynamics at the molecular level.

Wheat Ym2 originated from Aegilops sharonensis and confers resistance to soil-borne Wheat yellow mosaic virus infection to the roots.

Wheat yellow mosaic virus (WYMV) is a pathogen transmitted into its host's roots by the soil-borne vector Polymyxa graminis. Ym1 and Ym2 genes protect the host from the significant yield losses caused by the virus, but the mechanistic basis of these resistance genes remains poorly understood. Here, it has been shown that Ym1 and Ym2 act within the root either by hindering the initial movement of WYMV from the vector into the root and/or by suppressing viral multiplication. A mechanical inoculation experiment on the leaf revealed that the presence of Ym1 reduced viral infection incidence, rather than viral titer, while that of Ym2 was ineffective in the leaf. To understand the basis of the root specificity of the Ym2 product, the gene was isolated from bread wheat using a positional cloning approach. The candidate gene encodes a CC-NBS-LRR protein and it correlated allelic variation with respect to its sequence with the host's disease response. Ym2 (B37500) and its paralog (B35800) are found in the near-relatives, respectively, Aegilops sharonensis and Aegilops speltoides (a close relative of the donor of bread wheat's B genome), while both sequences, in a concatenated state, are present in several accessions of the latter species. Structural diversity in Ym2 has been generated via translocation and recombination between the two genes and enhanced by the formation of a chimeric gene resulting from an intralocus recombination event. The analysis has revealed how the Ym2 region has evolved during the polyploidization events leading to the creation of cultivated wheat.

Purification and ATPase Activity Measurement of Spiroplasma MreB.

Spiroplasma is a genus of wall-less helical bacteria with swimming motility unrelated to conventional types of bacterial motility machinery, such as flagella and pili. The swimming of Spiroplasma is suggested to be driven by five classes of MreB (MreB1-MreB5), which are members of the actin superfamily. In vitro studies of Spiroplasma MreBs have recently been conducted to evaluate their activities, such as ATPase, which is essential for the polymerization dynamics among classic actin superfamily proteins. In this chapter, we describe methods of purification and Pi release measurement of Spiroplasma MreBs using column chromatography and absorption spectroscopy with the molecular probe, 2-amino-6-mercapto-7-methylpurine riboside (MESG). Of note, the methods described here are applicable to other proteins that possess NTPase activity.

ATP-dependent polymerization dynamics of bacterial actin proteins involved in Spiroplasma swimming.

MreB is a bacterial protein belonging to the actin superfamily. This protein polymerizes into an antiparallel double-stranded filament that determines cell shape by maintaining cell wall synthesis. Spiroplasma eriocheiris, a helical wall-less bacterium, has five MreB homologous (SpeMreB1-5) that probably contribute to swimming motility. Here, we investigated the structure, ATPase activity and polymerization dynamics of SpeMreB3 and SpeMreB5. SpeMreB3 polymerized into a double-stranded filament with possible antiparallel polarity, while SpeMreB5 formed sheets which contained the antiparallel filament, upon nucleotide binding. SpeMreB3 showed slow Pi release owing to the lack of an amino acid motif conserved in the catalytic centre of MreB family proteins. Our SpeMreB3 crystal structures and analyses of SpeMreB3 and SpeMreB5 variants showed that the amino acid motif probably plays a role in eliminating a nucleophilic water proton during ATP hydrolysis. Sedimentation assays suggest that SpeMreB3 has a lower polymerization activity than SpeMreB5, though their polymerization dynamics are qualitatively similar to those of other actin superfamily proteins, in which pre-ATP hydrolysis and post-Pi release states are unfavourable for them to remain as filaments.

Magnetorheological Response for Magnetic Elastomers Containing Carbonyl Iron Particles Coated with Poly(methyl methacrylate).

The magnetorheological response for magnetic elastomers containing carbonyl iron (CI) particles with a diameter of 6.7 µm coated with poly(methyl methacrylate) (PMMA) was investigated to estimate the diameter of secondary particles from the amplitude of magnetorheological response. Fourier-transformed infrared spectroscopy revealed that the CI particles were coated with PMMA, and the thickness of the PMMA layer was determined to be 71 nm by density measurement. The change in the storage modulus for magnetic elastomers decreased by coating and it was scaled by the number density of CI particles as ΔG~N2.8. The diameter of secondary particle of CI particles coated with PMMA was calculated to be 8.4 µm. SEM images revealed that the CI particles coated with PMMA aggregated in the polyurethane matrix.

Sensitive and selective detection of prostate-specific antigen using a photonic crystal nanolaser.

The detection of low-concentration biomarkers is expected to facilitate the early diagnosis of severe diseases, including malignant tumors. Using photonic crystal nanolaser sensors, we detected prostate-specific antigen (PSA) from a concentration of 1 fM, which is difficult to detect by conventional enzyme-linked immunosorbent assay. The signal intensity and stability were improved by using a surfactant (i.e., ethanolamine). Even when a contaminant such as bovine serum albumin was mixed into the PSA sample, thereby increasing the concentration of the contaminant ten billion times, it was still possible to maintain a high level of detection.