Characterization of DmToll and DmToll7 homologue in Litopenaeus vannamei based on structure analysis.

Toll-like receptors (TLRs) are a family of pattern recognition receptors (PRRs) that recognize invading pathogens and activate downstream signaling pathways. The number of 10 Tolls is found in Litopenaeus vannamei but have not yet been identified as the corresponding Toll homologue of model animal. In this study, we predicted the three-dimensional (3D) structures of 10 LvTolls (LvToll1-10) with AlphaFold2 program. The per-residue local distance difference test (pLDDT) scores of LvTolls showed the predicted structure of LvTolls had high accuracy (pLDDT>70). By structural analysis, 3D structures of LvToll2 and LvToll3 had high similarity with Drosophila melanogaster Toll and Toll7, respectively. 3D structure of LvToll7 and LvToll10 were not similar to that of other LvTolls. Moreover, we also predicted that LvSpätzle4 had high structural similarity to DmSpätzle. There were 9 potential hydrogen bonds in LvToll2-LvSpätzle4 complex. Importantly, co-immunoprecipitation assay showed that LvToll2 could bind with LvSpätzle4. Collectively, this study provides new insight for researching invertebrate immunity by identifying the protein of model animal homologue.

Identification and characterization of a TLR4 homologue in Eriocheir sinensis based on structure analysis.

Toll-like receptor 4 (TLR4) plays an essential role in the activation of innate immunity by recognizing diverse pathogenic components of bacteria. Six Tolls were found in Eriocheir sinensis but have not yet been identified as mammalian TLR4 homolog. For this purpose, we predicted three-dimensional (3D) structures of EsTolls (EsToll1-6) with AlphaFold2. 3D structure of LRRs and TIR most had high accuracy (pLDDT >70). By structure analysis, 3D structures of EsToll6 had a high overlap with HsTLR4. Moreover, we also predicted potential 11 hydrogen bonds and 3 salt bridges in the 3D structure of EsToll6-EsML1 complex. 18 hydrogen bonds and 7 salt bridges were predicted in EsToll6-EsML2 complex. Co-immunoprecipitation assay showed that EsToll6 could interact with EsML1 and EsML2, respectively. Importantly, TAK242 (a mammalian TLR4-specific inhibitor) could inhibit the generation of ROS stimulated by lipopolysaccharides (LPS) in EsToll6-EsML2-overexpression Hela cells. Collectively, these results implied that EsToll6 was a mammalian TLR4 homolog and provided a new insight for researching mammalian homologs in invertebrates.

Shimia ponticola sp. nov., an aerobic anoxygenic photoheterotrophic bacterium, isolated from surface seawater in the South China Sea.

A Gram-stain-negative marine bacterium, designated as WX04T, was isolated from the South China Sea. The genome of strain WX04T contained a complete photosynthetic gene cluster and is the first identified photoheterotroph of the genus Shimia with high photochemical efficiency (Fv/Fm=0.705±0.010), indicating its diverse metabolic and growth strategies, and unique evolution in the genus Shimia. The genome size of strain WX04T is 3.78 Mbp, and the G+C content is 58.8 %. Its isolate formed pink colonies and the cells were non-flagellated and rod-shaped. Growth was observed at 15-35 °C (optimum, 30 °C), at pH 5.0-11.0 (optimum, pH 7.0) and in the presence of 3-5 % (w/v) NaCl (optimum, 3 %). Both catalase activity and oxidase activity were found to be negative. The 16S rRNA gene sequence analyses revealed that this isolate represents a novel species within the genus Shimia, sharing 96.8 and 95.6% sequence identities with Shimia aestuarii DSM 15283T and Shimia marina DSM 26895T, respectively. The respiratory quinone was ubiquinone-10 (100 %). The primary cellular fatty acids (>5 %) were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), C18 : 0,C18 : 1 ω7c 11-methyl and C10 : 0 3-OH. The dominant polar lipids of strain WX04T comprised phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol. The combined polyphasic data shows that strain WX04T is a novel species within the genus Shimia, which is proposed as Shimia ponticola sp. nov., and the type strain is WX04T (=KCTC 62628T=MCCC 1K02295T).

Numerical Analysis of Hydrogen Peroxide Addition and Oxygen-Enriched Methane Combustion.

Methane (CH4)/air lean combustion can be enhanced by increasing the concentration of the oxidizer, like oxygen (O2) enrichment, or adding a strong oxidant to the reactant. Hydrogen peroxide (H2O2) is a strong oxidizer that yields O2, steam, and appreciable heat after decomposition. This study numerically investigated and compared the effects of H2O2 and O2-enriched conditions on the adiabatic flame temperature, laminar burning velocity, flame thickness, and heat release rates of CH4/air combustion using the San Diego mechanism. The result showed that in fuel-lean conditions, the adiabatic flame temperature changed from H2O2 addition > O2-enriched scenario to O2-enriched scenario > H2O2 addition with increasing α. This transition temperature was not affected by the equivalence ratio. Adding H2O2 enhanced the laminar burning velocity of the CH4/air lean combustion more than the O2-enriched scenario. The thermal and chemical effects are quantified in various H2O2 additions, and it is found that the chemical effect has a noticeable contribution to the laminar burning velocity compared with the thermal effect, especially in higher H2O2 addition. Further, the laminar burning velocity had a quasi-linear correlation with (OH)max in the flame. The maximum heat release rate was observed at lower temperatures for H2O2 addition and higher temperatures for the O2-enriched scenario. The flame thickness was significantly reduced upon adding H2O2. Finally, the dominant reaction to the heat release rate changed from the reaction of CH3 + O ↔ CH2O + H in the CH4/air or O2-enriched scenario to the reaction of H2O2 + OH ↔ H2O + HO2 in the H2O2 addition scenario.

Diverse Subclade Differentiation Attributed to the Ubiquity of Prochlorococcus High-Light-Adapted Clade II.

Prochlorococcus is the key primary producer in marine ecosystems, and the high-light-adapted clade II (HLII) is the most abundant ecotype. However, the genomic and ecological basis of Prochlorococcus HLII in the marine environment has remained elusive. Here, we show that the ecologically coherent subclade differentiation of HLII corresponds to genomic and ecological characteristics on the basis of analyses of 31 different strains of HLII, including 12 novel isolates. Different subclades of HLII with different core and accessory genes were identified, and their distribution in the marine environment was explored using the TARA Oceans metagenome database. Three major subclade groups were identified, viz., the surface group (HLII-SG), the transition group (HLII-TG), and the deep group (HLII-DG). These subclade groups showed different temperature ranges and optima for distribution. In regression analyses, temperature and nutrient availability were identified as key factors affecting the distribution of HLII subclades. A 35% increase in the relative abundance of HLII-SG by the end of the 21st century was predicted under the Representative Concentration Pathway 8.5 scenario. Our results show that the ubiquity and distribution of Prochlorococcus HLII in the marine environment are associated with the differentiation of diverse subclades. These findings provide insights into the large-scale shifts in the Prochlorococcus community in response to future climate change. IMPORTANCEProchlorococcus is the most abundant oxygenic photosynthetic microorganism on Earth, and high-light-adapted clade II (HLII) is the dominant ecotype. However, the factors behind the dominance of HLII in the vast oligotrophic oceans are still unknown. Here, we identified three distinct groups of HLII subclades, viz., the surface group (HLII-SG), the transition group (HLII-TG), and the deep group (HLII-DG). We further demonstrated that the ecologically coherent subclade differentiation of HLII corresponds to genomic and ecological characteristics. Our study suggests that the differentiation of diverse subclades underlies the ubiquity and distribution of Prochlorococcus HLII in the marine environment and provides insights into the shifts in the Prochlorococcus community in response to future climate change.

A Novel Broad Host Range Phage Infecting Alteromonas.

Bacteriophages substantially contribute to bacterial mortality in the ocean and play critical roles in global biogeochemical processes. Alteromonas is a ubiquitous bacterial genus in global tropical and temperate waters, which can cross-protect marine cyanobacteria and thus has important ecological benefits. However, little is known about the biological and ecological features of Alteromonas phages (alterophages). Here, we describe a novel alterophage vB_AmeP-R8W (R8W), which belongs to the Autographiviridae family and infects the deep-clade Alteromonas mediterranea. R8W has an equidistant and icosahedral head (65 ± 1 nm in diameter) and a short tail (12 ± 2 nm in length). The genome size of R8W is 48,825 bp, with a G + C content of 40.55%. R8W possesses three putative auxiliary metabolic genes encoding proteins involved in nucleotide metabolism and DNA binding: thymidylate synthase, nucleoside triphosphate pyrophosphohydrolase, and PhoB. R8W has a rapid lytic cycle with a burst size of 88 plaque-forming units/cell. Notably, R8W has a wide host range, such that it can infect 35 Alteromonas strains; it exhibits a strong specificity for strains isolated from deep waters. R8W has two specific receptor binding proteins and a compatible holin-endolysin system, which contribute to its wide host range. The isolation of R8W will contribute to the understanding of alterophage evolution, as well as the phage-host interactions and ecological importance of alterophages.

Microbial transformation of distinct exogenous substrates into analogous composition of recalcitrant dissolved organic matter.

Oceanic dissolved organic matter (DOM) comprises a complex molecular mixture which is typically refractory and homogenous in the deep layers of the ocean. Though the refractory nature of deep-sea DOM is increasingly attributed to microbial metabolism, it remains unexplored whether ubiquitous microbial metabolism of distinct carbon substrates could lead to similar molecular composition of refractory DOM. Here, we conducted microbial incubation experiments using four typically bioavailable substrates (L-alanine, trehalose, sediment DOM extract, and diatom lysate) to investigate how exogenous substrates are transformed by a natural microbial assemblage. The results showed that although each-substrate-amendment induced different changes in the initial microbial assemblage and the amended substrates were almost depleted after 90 days of dark incubation, the bacterial community compositions became similar in all incubations on day 90. Correspondingly, revealed by ultra-high resolution mass spectrometry, molecular composition of DOM in all incubations became compositionally consistent with recalcitrant DOM and similar toward that of DOM from the deep-sea. These results indicate that while the composition of natural microbial communities can shift with substrate exposures, long-term microbial transformation of distinct substrates can ultimately lead to a similar refractory DOM composition. These findings provide an explanation for the homogeneous and refractory features of deep-sea DOM.

Pseudopontixanthobacter vadosimaris gen. nov., sp. nov., isolated from shallow sea near Kueishan Island.

A Gram-stain-negative and aerobic bacterial strain, designated as JL3514T, was isolated from surface water of the hydrothermal system around Kueishan Island. The isolate formed red colonies and cells were non-flagellated, rod-shaped and contained methanol-soluble pigments. Growth was observed at 10-50 °C (optimum, 30 °C), at pH 5.0-9.0 (optimum, pH 7.0) and in the presence of 0-9 % (w/v) NaCl (optimum, 2 %). Strain JL3514T was positive for catalase and weakly positive for oxidase. Results of 16S rRNA gene sequence analyses showed highest similarities to species in the family Erythrobacteraceae, namely Croceibacterium atlanticum (96.1 %), Pelagerythrobacter marensis (96.0 %), Tsuneonella rigui (96.0 %) and Altericroceibacterium xinjiangense (96.0 %). Phylogenetic analysis based on core gene sequences revealed that the isolate formed a distinct branch with the related species and it had a lower average amino acid identity value than the suggested threshold for genera boundaries. The major fatty acids (>5 %) were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C16 : 0, C17 : 1 ω6c, C14 : 0 2-OH and C12 : 0. The dominant polar lipids comprised diphosphatidylglycerol, sphingoglycolipid, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, glycolipid, two unidentified lipids and one unidentified phospholipid. The main respiratory quinones were ubiquinone-10 (95.7 %) and ubiquinone-9 (4.3 %). The DNA G+C content from the genome was 63.0 mol%. Based on the presented data, we consider strain JL3514T to represent a novel genus of the family Erythrobacteraceae, with the name Pseudopontixanthobacter vadosimaris gen. nov., sp. nov. The type strain is JL3514T (=KCTC 62623T=MCCC 1K03561T).

Yeosuana marina sp. nov., isolated from shallow-sea hydrothermal systems off Kueishantao Island.

A novel Gram-stain-negative, non-flagellated, non-motile, rod-shaped (0.4-0.6×1.8-2.5 µm), aerobic bacterial strain, designated JLT21T, was isolated from seawater of a shallow-sea hydrothermal system. Growth occurred with 0-4.0 % (w/v) NaCl (optimum, 2.0 % NaCl), at 8-45 °C (optimum, 25 °C) and at pH 3.0-10.0 (optimum, pH 7.0). Analysis of 16S rRNA gene sequences revealed that strain JLT21T showed the highest 16S rRNA gene sequence similarity to Gaetbulibacter aquiaggeris KEM-8T (97.2 %), Gaetbulibacter marinus IMCC1914T (96.9 %) and Yeosuana aromativorans GW1-1T (96.9 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain JLT21T clustered with Y. aromativorans GW1-1T. The predominant respiratory quinone of strain JLT21T was menaquinone-6 (MK-6). Polar lipids were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, three unidentified glycolipids and four unidentified lipids. The major fatty acids of strain JLT21T were iso-C15 : 0 (21.7 %), C16 : 1 ω6c/ω7c (11.5 %) and iso-C17 : 0 3-OH (10.9 %). The DNA G+C content of strain JLT21T was 32.6 %. On the basis of polyphasic analysis, strain JLT21T is considered to represent a novel species of the genus Yeosuana, for which the name Yeosuana marina sp. nov. is proposed. The type strain of Yeosuana marina is JLT21T (=CGMCC 1.15787T=JCM 31511T). The study helps us better understand the bacterial species in the shallow-sea hydrothermal system and their adaptations to the hydrothermal environment.

Paracoccus aurantiacus sp. nov., isolated from shallow-sea hydrothermal systems off Kueishantao Island.

An orange-pigmented, short-rod-shaped, aerobic and non-motile bacterial strain, designated TK008T, was isolated from the shallow-sea hydrothermal systems off Kueishantao Island in Taiwan, China, and it was studied by using a polyphasic taxonomic approach. Cells were Gram-stain-negative, and catalase- and oxidase-positive. Strain TK008T exhibited highest 16S rRNA gene sequence similarity of 97.1 % to Paracoccus pacificus F14T. The phylogenetic trees based on 16S rRNA gene sequences showed that strain TK008T was a member of the genus Paracoccus. Digital DNA-DNA hybridization values between strain TK008T and two closely related species (Paracoccus zhejiangensis and Paracoccus tegillarcae) were 20.6 and 20.9 %. The average nucleotide identity values of strain TK008T compared with P. zhejiangensis and P. tegillarcae were 75.2 and 74.6 % respectively. The major isoprenoid quinone was ubiquinone-10. The predominant fatty acids (>10 %) were summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, three unidentified phospholipids, two unidentified lipids and an unidentified glycolipid. The DNA G+C content of strain TK008T from genomic sequence data was 62.54 mol%. On the basis of polyphasic analysis, strain TK008T represents a novel species, for which the name Paracoccus aurantiacus sp. nov. is proposed. The type strain is TK008T (=CGMCC 1.13898T=JCM 33630T).

Ethanol flame synthesis of carbon nanotubes in deficient oxygen environments.

In this study, carbon nanotubes (CNTs) were synthesized using ethanol diffusion flames in a stagnation-flow system composed of an upper oxidizer duct and a lower liquid pool. In the experiments, a gaseous mixture of oxygen and nitrogen flowed from the upper oxidizer duct, and then impinged onto the vertically aligned ethanol pool to generate a planar and steady diffusion flame in a deficient oxygen environment. A nascent nickel mesh was used as the catalytic metal substrate to collect deposited materials. The effect of low oxygen concentration on the formation of CNTs was explored. The oxygen concentration significantly influenced the flame environment and thus the synthesized carbon products. Lowering the oxygen concentration increased the yield, diameter, and uniformity of CNTs. The optimal operating conditions for CNT synthesis were an oxygen concentration in the range of 15%-19%, a flame temperature in the range of 460 °C-870 °C, and a sampling position of 0.5-1 mm below the upper edge of the blue flame front. It is noteworthy that the concentration gradient of C2 species and CO governed the CNT growth directly. CNTs were successfully fabricated in regions with uniform C2 species and CO distributions.

Effects of Acoustic Modulation and Mixed Fuel on Flame Synthesis of Carbon Nanomaterials in an Atmospheric Environment.

In this study, methane-ethylene jet diffusion flames modulated by acoustic excitation in an atmospheric environment were used to investigate the effects of acoustic excitation frequency and mixed fuel on nanomaterial formation. Acoustic output power was maintained at a constant value of 10 W, while the acoustic excitation frequency was varied (f = 0-90 Hz). The results show that the flame could not be stabilized on the port when the ethylene volume concentration (ΩE) was less than 40% at f = 10 Hz, or when ΩE = 0% (i.e., pure methane) at f = 90 Hz. The reason for this is that the flame had a low intensity and was extinguished by the entrained air due to acoustic modulation. Without acoustic excitation (f = 0 Hz), the flame was comprised of a single-layer structure for all values of ΩE, and almost no carbon nanomaterials were synthesized. However, with acoustic excitation, a double-layer flame structure was generated for frequencies close to both the natural flickering frequency and the acoustically resonant frequency. This double-layer flame structure provided a favorable flame environment for the fabrication of carbon nanomaterials. Consequently, the synthesis of carbon nano-onions was significantly enhanced by acoustic excitation near both the natural flickering frequency and the acoustically resonant frequency. At f = 20 Hz (near the natural flickering frequency) for 0% ≤ ΩE ≤ 100%, a quantity of carbon nano-onions (CNOs) piled like bunches of grapes was obtained as a result of improved mixing of the fuel with ambient air. High-density CNOs were also produced at f = 70 Hz (close to the acoustically resonant frequency) for 40% ≤ ΩE ≤ 100%. Furthermore, carbon nanotubes (CNTs) were synthesized only at 80 Hz for ΩE = 0%. The suitable temperature range for the synthesis of CNTs was slightly higher than that for the formation of CNOs (about 600 °C for CNTs; 510-600 °C for CNOs).

Enhanced Synthesis of Carbon Nanomaterials Using Acoustically Excited Methane Diffusion Flames.

Acoustically modulated methane jet diffusion flames were used to enhance carbon nanostructure synthesis. A catalytic nickel substrate was employed to collect the deposit materials at sampling position z = 10 mm above the burner exit. The fabrication of carbon nano-onions (CNOs) and carbon nanotubes (CNTs) was significantly enhanced by acoustic excitation at frequencies near the natural flickering frequency (ƒ = 20 Hz) and near the acoustically resonant frequency (ƒ = 90 Hz), respectively. At these characteristic frequencies, flow mixing was markedly enhanced by acoustic excitation, and a flame structure with a bright slender core flame was generated, which provided a favorable flame environment for the growth of carbon nanomaterials. The production rate of CNOs was high at 20 Hz (near the natural flickering frequency), at which the gas temperature was about 680 °C. Additionally, a quantity of CNTs was obtained at 70-95 Hz, near the acoustically resonant frequency, at which the gas temperature was between 665 and 830 °C. However, no carbon nanomaterials were synthesized at other frequencies. The enhanced synthesis of CNOs and CNTs is attributed to the strong mixing of the fuel and oxidizer due to the acoustic excitation at resonant frequencies.

Analysis on controlling factors for the synthesis of carbon nanotubes and nano-onions in counterflow diffusion flames.

It is important to identify the dominant factors for governing the growth of carbon nanotubes (CNTs) and nano-onions (CNOs). A diffusion flame of a gas mixture of methane-ethylene was used as the carbon and heat sources and Ni as the catalyst for the synthesis of CNTs and CNOs. The effects of CH4/C2H4 ratio in the fuel side and oxygen concentration in the oxidizer side for counterflow diffusion flames were investigated. It was found that oxygen concentration can greatly affect the morphologies of synthesized products with a threshold of 30% distinguishing the formation of CNO or CNT. CNOs were fabricated at higher oxygen concentrations (30%, 40%, 50%), and CNTs were synthesized only at lower oxygen concentrations (21%, 30%). The fuel composition has minor effects on the morphologies except for the threshold value of oxygen concentration (30%). More carbon sources are required for the synthesis of CNOs than for CNTs, but the temperature requirements are similar (1140-1160 K for CNTs, 1070-1160 K for CNOs). The nanostructures were synthesized as long as the fuel concentration is sufficiently high regardless of the oxygen concentration. Higher fabrication tendency was found for ethylene as fuel to form nanostructures than for methane.

Two enzymes, TilS and HprT, can form a complex to function as a transcriptional activator for the cell division protease gene ftsH in Bacillus subtilis.

The FtsH protein is an ATP-dependent cytoplasmic membrane protease involved in the control of membrane protein quality, cell division and heat shock response in Bacillus subtilis and many other bacteria. TilS, the tRNA(Ile2) lysidine synthetase, is a tRNA-binding protein that can modify pre-tRNA(Ile2). HprT, the hypoxanthine-guanine phosphoribosyltransferase, is implicated in purine salvage. Both tilS and hprT are essential for cell viability of B. subtilis. In this report, by co-purification experiments and gel filtration analyses, we show that there is complex formation between co-expressed TilS and HprT. Electrophoretic mobility shift assays and in vitro transcription analyses demonstrated that the TilS/HprT complex functions as a specific DNA-binding protein that can stimulate ftsH transcription in vitro. Two regions located upstream of the ftsH promoter have been identified as the TilS/HprT-binding sites and shown to be required for TilS/HprT-dependent ftsH transcription in vitro and in vivo. Results from gel supershift assays support the notion that the TilS/HprT complex likely employs its distinct segments for interaction with these two distinct TilS/HprT-binding sites, respectively. In conclusion, we present the first evidence that bi-functional TilS and HprT can form a complex to function as a transcriptional activator to stimulate ftsH transcription.

Reexamining transcriptional regulation of the Bacillus subtilis htpX gene and the ykrK gene, encoding a novel type of transcriptional regulator, and redefining the YkrK operator.

HtpX is an integral cytoplasmic membrane metalloprotease well conserved in numerous bacteria. A recent study showed that expression of the Bacillus subtilis htpX gene is under dual negative control by Rok and a novel type of transcriptional regulator, YkrK. Here we report that expression of the B. subtilis htpX gene is strongly heat inducible. Contrary to the previous prediction, ykrK expression has been found to be not subject to autoregulation. We have identified the htpX promoter and the authentic ykrK promoter, which is also distinct from the previously predicted one. We have redefined a conserved inverted repeat sequence to be the YkrK operator, which is somewhat different from the previously proposed one. We provide evidence that YkrK is not a substrate of HtpX and that heat induction of htpX is not YkrK mediated. We have also found that the absence of FtsH or HtpX alone did not impair B. subtilis cell viability on LB agar plates at high temperature, whereas the absence of both FtsH and HtpX caused a severe growth defect under heat stress. This finding supports the notion that FtsH and HtpX may have partially overlapping functions in heat resistance. Finally, we show that htpX expression is subject to transient negative control by sigB under heat stress in a Rok- and YkrK-independent manner. Triple negative control of htpX expression at high temperature by rok, sigB, and ykrK may help cells to prevent uncontrolled and detrimental oversynthesis of the HtpX protease.

AdeR, a PucR-type transcription factor, activates expression of L-alanine dehydrogenase and is required for sporulation of Bacillus subtilis.

The Bacillus subtilis ald gene encodes L-alanine dehydrogenase, which catalyzes the NAD(+)-dependent deamination of L-alanine to pyruvate for the generation of energy and is required for normal sporulation. The transcription of ald is induced by alanine, but the mechanism underlying alanine induction remains unknown. Here we report that a gene (formerly yukF and now designated adeR) located upstream of ald is essential for the basal and alanine-inducible expression of ald. The disruption of the adeR gene caused a sporulation defect, whereas the complementation of an adeR mutation with an intact adeR gene restored the sporulation ability. adeR expression was not subject to autoregulation and alanine induction. Deletion and mutation analyses revealed that an inverted repeat, centered at position -74.5 relative to the transcriptional initiation site of ald, was required for ald expression and also likely served as a ρ-independent transcription terminator. Electrophoretic mobility shift assays showed that purified His-tagged AdeR was a specific DNA-binding protein and that this inverted repeat was required for AdeR binding. AdeR shows no significant amino acid sequence similarity to the known transcriptional activators of ald genes from other bacteria. AdeR appears to be a member of the PucR family of transcriptional regulators. Its orthologs of unknown function are present in some other Bacillus species. Collectively, these findings support the notion that AdeR is a transcriptional activator which mediates ald expression in response to alanine availability and is important for normal sporulation in B. subtilis.

PrcR, a PucR-type transcriptional activator, is essential for proline utilization and mediates proline-responsive expression of the proline utilization operon putBCP in Bacillus subtilis.

The soil bacterium Bacillus subtilis can utilize exogenous proline as a sole nitrogen or carbon source. The proline-inducible putBCP (formerly ycgMNO) operon encodes proteins responsible for proline uptake and two-step oxidation of proline to glutamate. We now report that a gene (formerly ycgP, now designated prcR) located downstream of the putBCP operon is essential for B. subtilis cells to utilize proline as a sole nitrogen or carbon source. Disruption of the prcR gene also abolished proline induction of putB transcription. prcR expression is not subject to autoregulation and proline induction. The PrcR protein shows no significant amino acid sequence similarity to the known transcriptional activators for proline utilization genes of other bacteria, but it does show partial amino acid sequence similarity to the transcriptional regulator PucR for the purine degradation genes of B. subtilis. PrcR orthologues of unknown function are present in some other Bacillus species. Primer-extension analysis suggests that both putB and prcR are transcribed by a σ(A)-dependent promoter. Deletion and mutation analysis revealed that an inverted repeat (5'-TTGTGG-N5-CCACAA-3') centred at position -76 relative to the transcriptional initiation site of putB is essential for putB expression. Electrophoretic mobility shift assays showed that the purified His-tagged PrcR was capable of binding specifically to this inverted repeat. Altogether, these results suggest that PrcR is a PucR-type transcriptional activator that mediates expression of the B. subtilis putBCP operon in response to proline availability.

Zebrafish HSC70 promoter to express carp muscle-specific creatine kinase for acclimation under cold condition.

Zebrafish (Danio rerio) is used as a model system for in vivo studies. To expand the research scope of physical, biochemical and physiological studies, a cold-tolerant model of zebrafish was developed. The common carp (Cyprinus carpio) muscle form of creatine kinase (CK, EC 2.7.3.2) can maintain enzymatic activity at a temperature of around 15°C. However, a cold-inducible promoter of zebrafish, hsc 70 (heat shock protein 70 cognate), is able to increase the expression of gene product by 9.8 fold at a temperature of 16°C. Therefore, the carp CK gene was promoted by hsc 70 and transfected into zebrafish embryos. Resulting transgenic zebrafish survived and could maintain its swimming behavior at 13°C, which was not possible with the wild-type zebrafish. The swimming distance of the transgenic fish was 42% greater than that of the wild type at 13°C. This new transgenic fish model is ideal for studies of ectothermal vertebrates in low-temperature environments.