The F region downstream of the mecI gene in the SCCmec element in hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) contains two bidirectionally overlapping open reading frames (ORFs), the fudoh ORF and the psm-mec ORF. The psm-mec ORF encodes a cytolysin, phenol-soluble modulin (PSM)-mec. Transformation of the F region into the Newman strain, which is a methicillin-sensitive S. aureus (MSSA) strain, or into the MW2 (USA400) and FRP3757 (USA300) strains, which are community-acquired MRSA (CA-MRSA) strains that lack the F region, attenuated their virulence in a mouse systemic infection model. Introducing the F region to these strains suppressed colony-spreading activity and PSMα production, and promoted biofilm formation. By producing mutations into the psm-mec ORF, we revealed that (i) both the transcription and translation products of the psm-mec ORF suppressed colony-spreading activity and promoted biofilm formation; and (ii) the transcription product of the psm-mec ORF, but not its translation product, decreased PSMα production. These findings suggest that both the psm-mec transcript, acting as a regulatory RNA, and the PSM-mec protein encoded by the gene on the mobile genetic element SCCmec regulate the virulence of Staphylococcus aureus.

Antifreeze proteins (AFPs) are found in organisms ranging from fish to bacteria, where they serve different functions to facilitate survival of their host. AFPs that protect freeze-intolerant fish and insects from internal ice growth bind to ice using a regular array of well-conserved residues/motifs. Less is known about the role of AFPs in freeze-tolerant species, which might be to beneficially alter the structure of ice in or around the host. Here we report the 0.95-Å high-resolution crystal structure of a 223-residue secreted AFP from the snow mold fungus Typhula ishikariensis. Its main structural element is an irregular β-helix with six loops of 18 or more residues that lies alongside an α-helix. β-Helices have independently evolved as AFPs on several occasions and seem ideally structured to bind to several planes of ice, including the basal plane. A novelty of the β-helical fold is the nonsequential arrangement of loops that places the N-and C termini inside the solenoid of β-helical coils. The ice-binding site (IBS), which could not be predicted from sequence or structure, was located by site-directed mutagenesis to the flattest surface of the protein. It is remarkable for its lack of regularity and its poor conservation in homologs from psychrophilic diatoms and bacteria and other fungi.X-ray crystallography | ice growth inhibition | thermal hysteresis

Femtosecond lasers have unique characteristics of ultrashort pulse width and extremely high peak intensity; however, one of the most important features of femtosecond laser processing is that strong absorption can be induced only at the focus position inside transparent materials due to nonlinear multiphoton absorption. This exclusive feature makes it possible to directly fabricate three-dimensional (3D) microfluidic devices in glass microchips by two methods: 3D internal modification using direct femtosecond laser writing followed by chemical wet etching (femtosecond laser-assisted etching, FLAE) and direct ablation of glass in water (water-assisted femtosecond laser drilling, WAFLD). Direct femtosecond laser writing also enables the integration of micromechanical, microelectronic, and microoptical components into the 3D microfluidic devices without stacking or bonding substrates. This paper gives a comprehensive review on the state-of-the-art femtosecond laser 3D micromachining for the fabrication of microfluidic, optofluidic, and electrofluidic devices. A new strategy (hybrid femtosecond laser processing) is also presented, in which FLAE is combined with femtosecond laser two-photon polymerization to realize a new type of biochip termed the ship-in-a-bottle biochip.

Antifreeze proteins (AFPs) are structurally diverse macromolecules that bind to ice crystals and inhibit their growth to protect the organism from injuries caused by freezing. An AFP identified from the Antarctic bacterium Colwellia sp. strain SLW05 (ColAFP) is homologous to AFPs from a wide variety of psychrophilic microorganisms. To understand the antifreeze function of ColAFP, we have characterized its antifreeze activity and determined the crystal structure of this protein. The recombinant ColAFP exhibited thermal hysteresis activity of approximately 4°C at a concentration of 0.14 mM, and induced rapid growth of ice crystals in the hexagonal direction. Fluorescence-based ice plane affinity analysis showed that ColAFP binds to multiple planes of ice, including the basal plane. These observations show that ColAFP is a hyperactive AFP. The crystal structure of ColAFP determined at 1.6 A resolution revealed an irregular b-helical structure, similar to known homologs. Mutational and molecular docking studies showed that ColAFP binds to ice through a compound ice-binding site (IBS) located at a flat surface of the b-helix and the adjoining loop region. The IBS of ColAFP lacks the repetitive sequences that are characteristic of hyperactive AFPs. These results suggest that ColAFP exerts antifreeze activity through a compound IBS that differs from the characteristic IBSs shared by other hyperactive AFPs. This study demonstrates a novel method for protection from freezing by AFPs in psychrophilic microorganisms. DatabaseStructural data for ColAFP have been submitted to the Protein Data Bank (PDB) under accession number 3WP9.

Snow mold fungus, Typhula ishikariensis, secretes seven antifreeze protein isoforms (denoted TisAFPs) that assist in the survival of the mold under snow cover. Here, the X-ray crystal structure of a hyperactive isoform, TisAFP8, at 1.0 Å resolution is presented. TisAFP8 folds into a right-handed β-helix accompanied with a long α-helix insertion. TisAFP8 exhibited significantly high antifreeze activity that is comparable with other hyperactive AFPs, despite its close structural and sequence similarity with the moderately active isoform TisAFP6. A series of mutations introduced into the putative ice-binding sites (IBSs) in the β-sheet and adjacent loop region reduced antifreeze activity. A double-mutant A20T/A212S, which comprises a hydrophobic patch between the β-sheet and loop region, caused the greatest depression of antifreeze activity of 75%, when compared with that of the wild-type protein. This shows that the loop region is involved in ice binding and hydrophobic residues play crucial functional roles. Additionally, bound waters around the β-sheet and loop region IBSs were organized into an ice-like network and can be divided into two groups that appear to mediate separately TisAFP and ice. The docking model of TisAFP8 with the basal plane via its loop region IBS reveals a better shape complementarity than that of TisAFP6. In conclusion, we present new insights into the ice-binding mechanism of TisAFP8 by showing that a higher hydrophobicity and better shape complementarity of its IBSs, especially the loop region, may render TisAFP8 hyperactive to ice binding.

We demonstrate the fabrication of three-dimensional (3-D) hollow microstructures embedded in photostructurable glass by a nonlinear multiphoton absorption process using a femtosecond (fs) laser. Fs laser direct writing followed by annealing and successive wet etching in dilute hydrofluoric (HF) acid solution resulted in the rapid manufacturing of microchips with 3-D hollow microstructures for the dynamic observation of living microorganisms and cells in fresh water. The embedded microchannel structure enables us to analyze the continuous motion of Euglena gracilis. A microchamber with a movable microneedle demonstrates its ability for the elucidation of the information transmission process in Pleurosira laevis. Such microchips, referred to as nano-aquariums realize the efficient and highly functional observation of microorganisms and cells.

Phormidium, a genus of filamentous cyanobacteria, forms endosymbiotic associations with seedling roots that accelerate the growth of the vegetable seedlings. Understanding the gliding mechanism of Phormidium will facilitate improved formation of this association and increased vegetable production. To observe the gliding movements, we fabricated various microfluidic chips termed nanoaquariums using a femtosecond (fs) laser. Direct fs laser writing, followed by annealing and successive wet etching in dilute hydrofluoric acid solution, can easily produce three-dimensional (3D) microfluidics with different structures embedded in a photostructurable glass. Using the fs laser, optical waveguides and filters were integrated with the microfluidic structures in the microchips, allowing the gliding mechanism to be more easily clarified. Using this apparatus, we found that CO(2) secreted from the seedling root attracts Phormidium in the presence of light, and determined the light intensity and specific wavelength necessary for gliding.

Internal modification of transparent materials such as glass can be carried out using multiphoton absorption induced by a femtosecond (fs) laser. The fs-laser modification followed by thermal treatment and successive chemical wet etching in a hydrofluoric (HF) acid solution forms three-dimensional (3D) hollow microstructures embedded in photosensitive glass. This technique is a powerful method for directly fabricating 3D microfluidic structures inside a photosensitive glass microchip. We used fabricated microchips, referred to as a nanoaquarium, for dynamic observations of living microorganisms. In addition, the present technique can also be used to form microoptical components such as micromirrors and microlenses inside the photosensitive glass, since the fabricated structures have optically flat surfaces. The integration of microfluidics and microoptical components in a single glass chip yields biophotonic microchips, in other words, optofluidics, which provide high sensitivity in absorption and fluorescence measurements of small volumes of liquid samples.Dynamic observation of microorganisms (Euglena gracilis) using nanoaquarium, which has 3D microfluidic structure, fabricated in a photosensitive glass microchip by femtosecond laser direct writing followed by thermal treatment and successive wet chemical etching.