A single-channel virtual receiving array using a time-reversal chaotic cavity.

Chaotic reverberation in a cavity, when coupled with time reversal acoustics, can be harnessed to build a perfect time-reversal mirror for transmitting and receiving highly focused sounds with a small number of transducers. In this article, a virtual receiving array, comprised of a single receiving transducer and a chaotic cavity, is developed based on time reversal processing of the reverberation inside the cavity. A prototype array, having 10 × 10 virtual receiving elements, is built and evaluated against a comparable physical array in terms of its localization and waveform reproduction capabilities. It turns out that the most crucial factor in the success of a virtual array is the ergodicity of its chaotic cavity, the exact mathematical expression for which is also derived. The virtual receiving array presented here may find some niche applications in reverberant environments, where a physical array turns out to be too costly or cumbersome to operate.

Semi-Biosynthetic Production of Surface-Binding Adhesive Antimicrobial Peptides Using Intein-Mediated Protein Ligation.

Microbial infections remain a global health concern, calling for the urgent need to implement effective prevention measures. Antimicrobial peptides (AMPs) have been extensively studied as potential antimicrobial coating agents. However, an efficient and economical method for AMP production is lacking. Here, we synthesized the direct coating adhesive AMP, NKC-DOPA5, composed of NKC, a potent AMP, and repeats of the adhesive amino acid 3,4-dihydroxyphenylalanine (DOPA) via an intein-mediated protein ligation strategy. NKC was expressed as a soluble fusion protein His-NKC-GyrA (HNG) in Escherichia coli, comprising an N-terminal 6× His-tag and a C-terminal Mxe GyrA intein. The HNG protein was efficiently produced in a 500-L fermenter, with a titer of 1.63 g/L. The NKC-thioester was released from the purified HNG fusion protein by thiol attack and subsequently ligated with chemically synthesized Cys-DOPA5. The ligated peptide His-NKC-Cys-DOPA5 was obtained at a yield of 88.7%. The purified His-NKC-Cys-DOPA5 possessed surface-binding and antimicrobial properties identical to those of the peptide obtained via solid-phase peptide synthesis. His-NKC-Cys-DOPA5 can be applied as a practical and functional antimicrobial coating to various materials, such as medical devices and home appliances.

Propagating acoustic waves on a culture substrate regulate the directional collective cell migration.

Collective cell migration plays a critical role in physiological and pathological processes such as development, wound healing, and metastasis. Numerous studies have demonstrated how various types of chemical, mechanical, and electrical cues dictate the collective migratory behaviors of cells. Although an acoustic cue can be advantageous because of its noninvasiveness and biocompatibility, cell migration in response to acoustic stimulation remains poorly understood. In this study, we developed a device that is able to apply surface acoustic waves to a cell culture substrate and investigated the effect of propagating acoustic waves on collective cell migration. The migration distance estimated at various wave intensities revealed that unidirectional cell migration was enhanced at a critical wave intensity and that it was suppressed as the intensity was further increased. The increased migration might be attributable to cell orientation alignment along the direction of the propagating wave, as characterized by nucleus shape. Thicker actin bundles indicative of a high traction force were observed in cells subjected to propagating acoustic waves at the critical intensity. Our device and technique can be useful for regulating cellular functions associated with cell migration.

Adhesive Antimicrobial Peptides Containing 3,4-Dihydroxy-L-Phenylalanine Residues for Direct One-Step Surface Coating.

Bacterial colonization and transmission via surfaces increase the risk of infection. In this study, we design and employ novel adhesive antimicrobial peptides to prevent bacterial contamination of surfaces. Repeats of 3,4-dihydroxy-L-phenylalanine (DOPA) were added to the C-terminus of NKC, a potent synthetic antimicrobial peptide, and the adhesiveness and antibacterial properties of the resulting peptides are evaluated. The peptide is successfully immobilized on polystyrene, titanium, and polydimethylsiloxane surfaces within 10 min in a one-step coating process with no prior surface functionalization. The antibacterial effectiveness of the NKC-DOPA5-coated polystyrene, titanium, and polydimethylsiloxane surfaces is confirmed by complete inhibition of the growth of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus within 2 h. The stability of the peptide coated on the substrate surface is maintained for 84 days, as confirmed by its bactericidal activity. Additionally, the NKC-DOPA5-coated polystyrene, titanium, and polydimethylsiloxane surfaces show no cytotoxicity toward the human keratinocyte cell line HaCaT. The antimicrobial properties of the peptide-coated surfaces are confirmed in a subcutaneous implantation animal model. The adhesive antimicrobial peptide developed in this study exhibits potential as an antimicrobial surface-coating agent for efficiently killing a broad spectrum of bacteria on contact.

Optical dosimeter for selective retinal therapy based on multi-port fiber-optic interferometry.

Selective retinal therapy (SRT) employs a micro-second short-pulse lasers to induce localized destruction of the targeted retinal structures with a pulse duration and power aimed at minimal damage to other healthy retinal cells. SRT has demonstrated a great promise in the treatment of retinal diseases, but pulse energy thresholds for effective SRT procedures should be determined precisely and in real time, as the thresholds could vary with disease status and patients. In this study, we present the use of a multi-port fiber-based interferometer (MFI) for highly sensitive real-time SRT monitoring. We exploit distinct phase differences among the fiber ports in the MFI to quantitatively measure localized fluctuations of complex-valued information during the SRT procedure. We evaluate several metrics that can be computed from the full complex-valued information and demonstrate that the complex contour integration is highly sensitive and most correlative to pulse energies, acoustic outputs, and cell deaths. The validity of our method was demonstrated on excised porcine retinas, with a sensitivity and specificity of 0.92 and 0.88, respectively, as compared with the results from a cell viability assay.

Lack of the Bacterial Phytochrome Protein Decreases Deinococcus radiodurans Resistance to Mitomycin C.

Deinococcus radiodurans known for its extraordinary resistance to ionizing radiation contains bacterial phytochrome (BphP), a member of the family of red/far-red light-sensing proteins. In this study, we constructed a bphP mutant strain (ΔbphP) to investigate the role of D. radiodurans BphP (DrBphP) in the DNA damage response. When cells were incubated under light and dark conditions following exposure to DNA damaging agents, such as γ- and UV-radiation and mitomycin C (MMC), no significant difference in cell survival was observed between the wild-type D. radiodurans strain (WT) and ΔbphP. However, when continuously exposed to MMC under light conditions, the WT strain notably exhibited increased survival compared to cells grown in the dark. The increased survival was not observed in the ΔbphP strain. These results are indicative of the protective role of light-activated DrBphP in the presence of MMC. Site-directed mutagenesis revealed that the conserved amino acids Cys-24 and His-532 involved in chromophore binding and signal transduction, respectively, were essential for the protective function of DrBphP. Inactivation of the cognate response regulator (RR; DrBphR) of DrBphP increased MMC resistance in the dark. In trans complementation of the bphP bphR double mutant strain (ΔbphPR) with DrBphR decreased MMC resistance. Considering that DrBphP acts as a light-activated phosphatase that dephosphorylates DrBphR, it appears that phosphorylated DrBphR exerts a negative effect on cell survival in the presence of MMC. DrBphP overexpression resulted in an increase in MMC resistance of ΔbphPR, implying that other RRs might be involved in the DrBphP-mediated signaling pathway. A mutant lacking the dr_0781 gene (Δdr_0781) demonstrated the same MMC phenotype as ΔbphR. Survival was further increased in the bphR dr_0781 double mutant strain compared to each single mutant ΔbphR or Δdr_0781, suggesting that DR_0781 is also involved in the DrBphP-dependent MMC sensitivity. This study uncovered a previously unknown phenomenon of red/far-red light-dependent DNA damage survival mediated by BphP by identifying the conditions under which DrBphP exhibits a fitness advantage.

Deinococcus radioresistens sp. nov., a UV and gamma radiation-resistant bacterium isolated from mountain soil.

Two Gram-negative, non-motile, short rod-shaped bacterial strains, designated as 8A(T) and 28A, were isolated from Mount Deogyusan, Jeonbuk Province, South Korea. The isolates were analyzed by a polyphasic approach, revealing variations in their phenotypic characters but high DNA-DNA hybridisation values reciprocally, confirming that they belong to the same species. Both the isolates also showed a high resistance to UV compared with Deinococcus radiodurans, and a gamma-radiation resistance similar to other members of the genus Deinococcus. Phylogenetic analysis with the 16S rRNA gene sequences of closely related species indicated their similarities were below 97 %. Chemotaxonomic data showed the most abundant fatty acids to be C16:1ω7c and C16:0. The strains can be distinguished from closely related species by the production of esterase (C4) and α-galactosidase, and by their ability to assimilate L-alanine, L-histidine and N-acetyl-D-glucosamine. Based on the phenotypic, phylogenetic, and chemotaxonomic data, the isolates represent a novel species of the genus Deinococcus, for which the name Deinococcus radioresistens sp. nov. is proposed. The type strain is 8A(T) (KEMB 9004-109(T) = JCM 19777(T)), and a second strain is 28A (KEMB 9004-113 = JCM 19778).

Deinococcus puniceus sp. nov., a bacterium isolated from soil-irradiated gamma radiation.

A Gram-positive, coccus-shaped, crimson-color-pigmented bacterium was isolated from soil irradiated with 5 kGy gamma radiation and was designated strain DY1(T). Cells showed growth at 10-30 °C and pH 7-11 and were oxidase-negative and catalase-positive. Phylogenetic analyses of the 16S rRNA gene showed that the strain DY1(T) belonged to the genus Deinococcus with sequence similarities to Deinococcus aquatilis CCUG 53370(T) (96.2 %) and Deinococcus navajonensis KR-114(T) (94.1 %). Strain DY1(T) showed low level of DNA relatedness with D. aquatilis CCUG 53370(T) (41.3 ± 3.9 %). The DNA G + C content of DY1(T) was 58.7 mol%. Predominant fatty acids were summed feature 3 (C16:1 ω7c/ω6c), C16:0, and C17:0. The major amino acids were D-alanine, L-glutamic acid, glycine, and L-ornithine in the peptidoglycan. The major polar lipids were unknown phosphoglycolipids (PGL). Strain DY1(T) has resistance to gamma radiation and was found to be a novel species. Therefore, the strain was designated as DY1(T) (=KCTC 33027(T) = JCM 18576(T)), and the name Deinococcus puniceus sp. nov. is herein proposed.

Spirosoma radiotolerans sp. nov., a gamma-radiation-resistant bacterium isolated from gamma ray-irradiated soil.

A Gram-negative, short-rod-shaped bacterial strain with gliding motility, designated as DG5A(T), was isolated from a rice field soil in South Korea. Phylogenic analysis using 16S rRNA gene sequence of the new isolate showed that strain DG5A(T) belong to the genus Spirosoma in the family Spirosomaceae, and the highest sequence similarities were 95.5 % with Spirosoma linguale DSM 74(T), 93.4 % with Spirosoma rigui WPCB118(T), 92.8 % with Spirosoma luteum SPM-10(T), 92.7 % with Spirosoma spitsbergense SPM-9(T), and 91.9 % with Spirosoma panaciterrae Gsoil 1519(T). Strain DG5A(T) revealed resistance to gamma and UV radiation. Chemotaxonomic data showed that the most abundant fatty acids were summed feature C(16:1) ω7c/C(16:1) ω6c (36.90 %), C(16:1) ω5c (29.55 %), and iso-C(15:0) (14.78 %), and the major polar lipid was phosphatidylethanolamine (PE). The DNA G+C content of strain DG5A(T) was 49.1 mol%. Together, the phenotypic, phylogenetic, and chemotaxonomic data supported that strain DG5A(T) presents a novel species of the genus Spirosoma, for which the name Spirosoma radiotolerans sp. nov., is proposed. The type strain is DG5A(T) (=KCTC 32455(T) = JCM19447(T)).

Transcriptome analysis of salt-stressed Deinococcus radiodurans and characterization of salt-sensitive mutants.

Deinococcus radiodurans is a bacterium best known for its extreme resistance to high levels of ionizing radiation. Gene expression profiles of D. radiodurans exposed to 0.3 M NaCl revealed that at least 389 genes were induced and 415 were repressed by twofold or more. A general down-regulation of the central metabolic pathways and a strong decrease of nrd gene expression, which encodes proteins necessary for DNA synthesis, likely reflect the growth retardation induced by NaCl stress. The expression of rsbRSTX, which encodes sigma B (σ(B)) activity regulators, was also reduced by NaCl stress even though D. radiodurans does not have σ(B). The mutation of rsbX (drB0027) decreased the tolerance of D. radiodurans to NaCl, suggesting the possible role of the Rsb module in NaCl response. On the other hand, NaCl stress activated genes associated with osmoprotectant accumulation: the pstSCAB operon, which encodes a high affinity phosphate transporter, and DRA0135 and DR1438, which are components of transporters of glycine betaine and trehalose. Survival analysis of mutant strains lacking DR0392 (membrane-binding protein) and DR1115 (S-layer protein), whose expressions were highly activated by NaCl, showed a reduction in NaCl tolerance. In addition, the Δdr0392 strain showed sensitivity to γ-irradiation compared to the wild type. These results suggest that DR0392 plays a role in the resistance of D. radiodurans to NaCl and γ-irradiation.

Comparative survival analysis of 12 histidine kinase mutants of Deinococcus radiodurans after exposure to DNA-damaging agents.

Bacteria are able to adapt to changes in the environment using two-component signal transduction systems (TCSs) composed of a histidine kinase (HK) and a response regulator (RR). Deinococcus radiodurans, one of the most resistant organisms to ionizing radiation, has 20 putative HKs and 25 putative RRs. In this study, we constructed 12 D. radiodurans mutant strains lacking a gene encoding a HK and surveyed their resistance to γ-radiation, UV-B radiation (302 nm), mitomycin C (MMC), and H(2)O(2). Five (dr0860 (-), dr1174 (-), dr1556 (-), dr2244 (-), and dr2419 (-)) of the 12 mutant strains showed at least a one-log cycle reduction in γ-radiation resistance. The mutations (1) dr1174, dr1227, and dr2244 and (2) dr0860, dr2416, and dr2419 caused decreases in resistance to UV radiation and MMC, respectively. Only the dr2416 and dr2419 mutant strains showed higher sensitivity to H(2)O(2) than the wild-type. Reductions in the resistance to γ-radiation and H(2)O(2), but not to UV and MMC, were observed in the absence of DR2415, which seems to be a cognate RR of DR2416. This result suggests that DR2415/DR2416 (DrtR/S: DNA damage response TCS) may be another TCS responsible for the extreme resistance of D. radiodurans to DNA-damaging agents.

Expression and bioconversion of recombinant m- and p-hydroxybenzoate hydroxylases from a novel moderate halophile, Chromohalobacter sp.

p-Hydroxybenzoate hydroxylase (pobA) and m-hydroxybenzoate hydroxylase (mobA) genes, from the moderate halophile Chromohalobacter sp. HS-2, were expressed and characterized. Solubilities of overexpressed recombinant MobA and PobA were enhanced by the induction of the heat-shock proteins DnaJ and DnaK. Each MobA and PobA maintained stable activity under high NaCl concentrations. V (max) and K (m) values for MobA with m-hydroxybenzoate were 70 µmol min(-1) mg(-1) protein and 81 µM, respectively. Similarly, those of PobA with p-hydroxybenzoate as substrate were 5 µmol min(-1) mg(-1) protein and 129 µM, respectively. The Escherichia coli expression system, including induction of heat shock proteins, was used to convert hydroxybenzoates into protocatechuate (3,4-dihydroxybenzoate) and revealed that resting cells harboring mobA converted 15 mM m-hydroxybenzoate to 15 mM protocatechuate while those harboring pobA converted 50 mM p-hydroxybenzoate to 35 mM protocatechuate at 30 °C, respectively.

Pigment-based whole-cell biosensor system for cadmium detection using genetically engineered Deinococcus radiodurans.

In this study, a colorimetric whole-cell biosensor for cadmium (Cd) was designed using a genetically engineered red pigment producing bacterium, Deinococcus radiodurans. Based on the previous microarray data, putative promoter regions of highly Cd-inducible genes (DR_0070, DR_0659, DR_0745, and DR_2626) were screened and used for construction of lacZ reporter gene cassettes. The resultant reporter cassettes were introduced into D. radiodurans R1 to evaluate promoter activity and specificity. Among the promoters, the one derived from DR_0659 showed the highest specificity, sensitivity, and activity in response to Cd. The Cd-inducible activity was retained in the 393-bp deletion fragment (P0659-1) of the P0569 promoter, but the expression pattern of the putative promoter fragments inferred its complex regulation. The detection range was from 10 to 1 mM of Cd. The LacZ expression was increased up to 100 µM of Cd, but sharply decreased at higher concentrations. For macroscopic detection, the sensor plasmid (pRADI-P0659-1) containing crtI as a reporter gene under the control of P0659-1 was introduced into a crtI-deleted mutant strain of D. radiodurans (KDH018). The color of this sensor strain (KDH081) changed from light yellow to red by the addition of Cd and had no significant response to other metals. Color change by the red pigment synthesis could be clearly recognized in a day with the naked eye and the detection range was from 50 nM to 1 mM of Cd. These results indicate that genetically engineered D. radiodurans (KDH081) can be used to monitor the presence of Cd macroscopically.

Genome-wide response of Deinococcus radiodurans on cadmium toxicity.

Deinococcus radiodurans is extremely resistant to various genotoxic conditions and chemicals. In this study, we characterized the effect of a sublethal concentration (100 microM) of cadmium (Cd) on D. radiodurans using a whole-genome DNA microarray. Time-course global gene expression profiling showed that 1,505 genes out of 3,116 total ORFs were differentially expressed more than 2-fold in response to Cd treatment for at least one timepoint. The majority of the upregulated genes are related to iron uptake, cysteine biosynthesis, protein disulfide stress, and various types of DNA repair systems. The enhanced upregulation of genes involved in cysteine biosynthesis and disulfide stress indicate that Cd has a high affinity for sulfur compounds. Provocation of iron deficiency and growth resumption of Cd-treated cells by iron supplementation also indicates that CdS forms in iron-sulfur-containing proteins such as the [Fe-S] cluster. Induction of base excision, mismatch, and recombinational repair systems indicates that various types of DNA damage, especially base excision, were enhanced by Cd. Exposure to sublethal Cd stress reduces the growth rate, and many of the downregulated genes are related to cell growth, including biosynthesis of cell membrane, translation, and transcription. The differential expression of 52 regulatory genes suggests a dynamic operation of complex regulatory networks by Cd-induced stress. These results demonstrate the effect of Cd exposure on D. radiodurans and how the related genes are expressed by this stress.

Comparison of the genomes of deinococcal species using oligonucleotide microarrays.

The bacterium Deinococcus radiodurans is one of the most resistant organisms to ionizing radiation and other DNAdamaging agents. Although, at present, 30 Deinococcus species have been identified, the whole-genome sequences of most species remain unknown, with the exception of D. radiodurans (DRD), D. geothermalis, and D. deserti. In this study, comparative genomic hybridization (CGH) microarray analysis of three Deinococcus species, D. radiopugnans (DRP), D. proteolyticus (DPL), and D. radiophilus (DRPH), was performed using oligonucleotide arrays based on DRD. Approximately 28%, 14%, and 15% of 3,128 open reading frames (ORFs) of DRD were absent in the genomes of DRP, DPL, and DRPH, respectively. In addition, 162 DRD ORFs were absent in all three species. The absence of 17 randomly selected ORFs was confirmed by a Southern blot. Functional classification showed that the absent genes spanned a variety of functional categories: some genes involved in amino acid biosynthesis, cell envelope, cellular processes, central intermediary metabolism, and DNA metabolism were not present in any of the three deinococcal species tested. Finally, comparative genomic data showed that 120 genes were Deinococcus-specific, not the 230 reported previously. Specifically, ddrD, ddrO, and ddrH genes, previously identified as Deinococcus-specific, were not present in DRP, DPL, or DRPH, suggesting that only a portion of ddr genes are shared by all members of the genus Deinococcus.