Mercury (II) sensor based on monitoring dissociation rate of the trans-acting factor MerR from cis-element by surface plasmon resonance.

Transcriptional switches regulate gene expression in response to environmental changes surrounding cell. Many studies have focused on two fundamentally different models of transcriptional control by bacterial metalloregulatory protein. Distortion of the DNA fragment including cis-element, to which the trans-acting factor MerR binds, is accepted as the mechanism of gene expression regulation by Hg (II) while, in cases of the other trans-acting factors ArsR and CadC, events of association to and dissociation from cis-element are known to control transcription in response to As (III) and Cd (II), respectively. In this study, interactions between green-fluorescent-protein-tagged trans-acting factor and immobilized cis-element were analyzed on solid surface. Fluorescent measurements and surface plasmon resonance (SPR) responses revealed that although the equilibrium dissociation constant (KD) was much lower in MerR than in ArsR and CadC, the dissociation rate of MerR from DNA increased in response to Hg (II) at concentrations of 5-10(4) µg l(-1). These results firstly demonstrate an increase of KD between MerR and its recognition site in DNA by Hg (II), and possibility of rapid Hg (II) quantification with the low detection limit (5 µg l(-1)) and the high dynamic range (10(1)-10(4) µg l(-1)).

3-Hydroxybutyrate oligomer hydrolase and 3-hydroxybutyrate dehydrogenase participate in intracellular polyhydroxybutyrate and polyhydroxyvalerate degradation in Paracoccus denitrificans.

Genes encoding 3-hydroxybutyrate oligomer hydrolase (PhaZc) and 3-hydroxybutyrate dehydrogenase (Hbd) were isolated from Paracoccus denitrificans. PhaZc and Hbd were overproduced as His-tagged proteins in Escherichia coli and purified by affinity and gel filtration chromatography. Purified His-tagged proteins had molecular masses of 31 kDa and 120 kDa (a tetramer of 29-kDa subunits). The His-tagged PhaZc hydrolyzed not only 3-hydroxybutyrate oligomers but also 3-hydroxyvalerate oligomers. The His-tagged Hbd catalyzed the dehydrogenation of 3-hydroxyvalerate as well as 3-hydroxybutyrate. When both enzymes were included in the same enzymatic reaction system with 3-hydroxyvalerate dimer, sequential reactions occurred, suggesting that PhaZc and Hbd play an important role in the intracellular degradation of poly(3-hydroxyvalerate). When the phaZc gene was disrupted in P. denitrificans by insertional inactivation, the mutant strain lost PhaZc activity. When the phaZc-disrupted P. denitrificans was complemented with phaZc, PhaZc activity was restored. These results suggest that P. denitrificans carries a single phaZc gene. Disruption of the phaZc gene in P. denitrificans affected the degradation rate of PHA.

Acyclic carotenoid and cyclic apocarotenoid cleavage by an orthologue of lignostilbene-α,ß-dioxygenase in Rhodopseudomonas palustris.

Carotenoid cleavage oxygenases catalyse formation of apocarotenoids and the precursors of phytohormones, abscisic acid and strigolactones through oxidative cleavage at specific double bonds of carotenoids. A gene encoding a presumed bacterial oxygenase homologous to lignostilbene-α,ß-dioxygenases has been found in the genome of Rhodopseudomonas palustris. By analysing apocarotenoids in recombinant Escherichia coli strains, it was found that the presumed oxygenase catalyses the 15,15' double bond cleavage of lycopene and neurosporene. Cell lysate containing the recombinant protein cleaved all-trans-ß-apo-8'-carotenal at the 15,15' double bond into retinal and apo-8',15'-apocarotene-dial. These data demonstrate for the first time that the orthologue of lignostilbene-α,ß-dioxygenase found in the carotenogenic phototrophic bacterium has the 15,15' double bond cleavage activity towards both the acyclic carotenoids and cyclic apocarotenoid.

Thermoresponsive magnetic nano-biosensors for rapid measurements of inorganic arsenic and cadmium.

Green fluorescent protein-tagged sensor proteins, ArsR-GFP and CadC-GFP, have been produced as biosensors for simple and low-cost quantification of As(III) or Cd(II). In this study, the sensor protein-promoter DNA complexes were reconstructed on the surfaces of magnetic particles of different sizes. After the surface modification all the particles could be attracted by magnets, and released different amounts of GFP-tagged protein, according to the metal concentrations within 5 min, which caused significant increases in fluorescence. A detection limit of 1 µg/L for As(III) and Cd(II) in purified water was obtained only with the nanoparticles exhibiting enough magnetization after heat treatment for 1 min. Therefore, thermoresponsive magnetic nano-biosensors offer great advantages of rapidity and sensitivity for the measurement of the toxic metals in drinking water.

Fluorescent bioassays for toxic metals in milk and yoghurt.

BACKGROUND: From a human health viewpoint, contaminated milk and its products could be a source of long-term exposure to toxic metals. Simple, inexpensive, and on-site assays would enable constant monitoring of their contents. Bioassays that can measure toxic metals in milk or yoghurt might reduce the risk. For this purpose, the green fluorescent protein (GFP)-tagged trans factors, ArsR-GFP and CadC-GFP, together with their cis elements were used to develop such bioassays. RESULTS: ArsR-GFP or CadC-GFP, which binds either toxic metal or DNA fragment including cis element, was directly mixed with cow's milk or yoghurt within a neutral pH range. The fluorescence of GFP, which is reflected by the association/dissociation ratio between cis element and trans factor, significantly changed with increasing externally added As (III) or Cd (II) whereas smaller responses to externally added Pb (II) and Zn (II) were found. Preparation and dilution of whey fraction at low pH were essential to intrinsic zinc quantification using CadC-GFP. Using the extraction procedure and bioassay, intrinsic Zn (II) concentrations ranging from 1.4 to 4.8 mg/l for milk brands and from 1.2 to 2.9 mg/kg for yoghurt brands were determined, which correlated to those determined using inductively coupled plasma atomic emission spectroscopy. CONCLUSIONS: GFP-tagged bacterial trans factors and cis elements can work in the neutralized whole composition and diluted whey fraction of milk and yoghurt. The feature of regulatory elements is advantageous for establishment of simple and rapid assays of toxic metals in dairy products.

Solid phase biosensors for arsenic or cadmium composed of A trans factor and cis element complex.

The presence of toxic metals in drinking water has hazardous effects on human health. This study was conducted to develop GFP-based-metal-binding biosensors for on-site assay of toxic metal ions. GFP-tagged ArsR and CadC proteins bound to a cis element, and lost the capability of binding to it in their As- and Cd-binding conformational states, respectively. Water samples containing toxic metals were incubated on a complex of GFP-tagged ArsR or CadC and cis element which was immobilized on a solid surface. Metal concentrations were quantified with fluorescence intensity of the metal-binding states released from the cis element. Fluorescence intensity obtained with the assay significantly increased with increasing concentrations of toxic metals. Detection limits of 1 µg/L for Cd(II) and 5 µg/L for As(III) in purified water and 10 µg/L for Cd(II) and As(III) in tap water and bottled mineral water were achieved by measurement with a battery-powered portable fluorometer after 15-min and 30-min incubation, respectively. A complex of freeze dried GFP-tagged ArsR or CadC binding to cis element was stable at 4 °C and responded to 5 µg/L As(III) or Cd(II). The solid phase biosensors are sensitive, less time-consuming, portable, and could offer a protocol for on-site evaluation of the toxic metals in drinking water.

Monitoring of environmental arsenic by cultures of the photosynthetic bacterial sensor illuminated with a near-infrared light emitting diode array.

Recombinant Rhodopseudomonas palustris, harboring the carotenoid-metabolizing gene crtI (CrtIBS), and whose color changes from greenish yellow to red in response to inorganic As(III), was cultured in transparent microplate wells illuminated with a light emitting diode (LED) array. The cells were seen to grow better under near-infrared light, when compared with cells illuminated with blue or green LEDs. The absorbance ratio of 525 to 425 nm after cultivation for 24 h, which reflects red carotenoid accumulation, increased with an increase in As(III) concentrations. The detection limit of cultures illuminated with near-infrared LED was 5 microgram/l, which was equivalent to that of cultures in test tubes illuminated with an incandescent lamp. A near-infrared LED array, in combination with a microplate, enabled the simultaneous handling of multiple cultures, including CrtIBS and a control strain, for normalization by the illumination of those with equal photon flux densities. Thus, the introduction of a near-infrared LED array to the assay is advantageous for the monitoring of arsenic in natural water samples that may contain a number of unknown factors and, therefore, need normalization of the reporter event.

Application of fluorescent protein-tagged trans factors and immobilized cis elements to monitoring of toxic metals based on in vitro protein-DNA interactions.

Environmental toxic metals cause serious global public health problems. On-site monitoring protects people from exposure to such harmful elements. In this study, the bacterial transcriptional switches were applied to monitoring of toxic metals. ArsR and CadC, trans factors of Escherichia coli and Staphylococcus aureus, were fused to GFP. The fusion proteins, ArsR-GFP and CadC-GFP, associated with cis elements, P(ars)-O(ars) and P(cad)-O(cad), respectively and dissociated from those upon recognition of As(III) or Pb/Cd. Cell lysates containing ArsR-GFP were pre-incubated with As(III) standard solutions for 15 min and loaded into P(ars)-O(ars)-immobilized microplate wells. Cell lysates containing CadC-GFP were pre-incubated with Pb or Cd solutions and loaded into P(cad)-O(cad)-immobilized wells. The cell lysates were incubated for 15 min and removed from the wells. Fluorescence intensity in the wells dose-dependently decreased in response to As(III) up to 200 µg/l or Pb/Cd up to 100 µg/l. Detection limits were 10 µg/l for As(III) 10 µg/l for Cd, and 20 µg/l for Pb with a microplate fluororeader, whereas 5.0 µg/l for As(III), 1.0 µg/l for Cd, and 10 µg/l for Pb with a handheld fluorometer. This method was available to detect Pb/Cd or As(III) in water containing soil extracts. This is the first demonstration of a simple and rapid fluorometry to detect analytes based on in vitro interaction between a cis element and a trans factor.

Genetic replacement of tesB with PTE1 affects chain-length proportions of 3-hydroxyalkanoic acids produced through ß-oxidation of oleic acid in Escherichia coli.

Acyl-CoA thioesterase II (TesB), which catalyzes hydrolysis of acyl-CoAs to free fatty acids and CoA, is involved in 3-hydroxyalkanoic acid production in Escherichia coli. Effects of genetic replacement of tesB with Saccharomyces cerevisiae acyl-CoA thioesterase gene PTE1 on 3-hydroxyalkanoic acid production from oleic acid through ß-oxidation were examined. Kinetic analyses using ß-oxidation intermediates showed that hydrolyses of C4-acyl substrates are more efficient by PTE1 than by TesB. Deletion of tesB in E. coli decreased 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, and hexanoic acid in medium after cultivation with oleic acid as a sole carbon source. Hexanoic acid concentration was much lower than those of 3-hydroxyacids. In genetic complementation of tesB deletion, use of PTE1, instead of tesB, affected proportions of the 3-hydroxyalkanoic acids. Proportion of 3-hydroxybutyric acid was higher in a PTE1-complemented strain than in a tesB-complemented strain, while proportions of 3-hydroxyhexanoic acid and 3-hydroxyoctanoic acid markedly increased in the tesB-complemented strain. Proportion of 3-hydroxyoctanoic acid did not significantly increase in the PTE1-complemented strain. These data indicate possibilities of 3-hydroxyalkanoic acid production from oleic acid through ß-oxidation and customization of their chain-length proportions by genetic replacement of tesB with a gene encoding acyl-CoA thioesterase with a different kinetic property.

Sensitive fluorescent microplate bioassay using recombinant Escherichia coli with multiple promoter-reporter units in tandem for detection of arsenic.

Genetically modified bacterial biosensors can detect specific environmental compounds. Here, we attempted to establish a fluorescent microplate method to detect arsenic using recombinant Escherichia coli cells transformed with plasmids harboring three tandem copies of the ars promoter/operator-the gene for green fluorescent protein (gfp). In the biosensors, one copy of arsR, whose transcription is autoregulated by the ars promoter/operator and ArsR in the genome of E. coli, was placed in trans in another plasmid under the control of isopropyl-1-thio-beta-D-galactopyranoside-inducible promoter. First, this manipulation enabled regulation of the arsR expression at an adequate level. Second, the copy number of reporter unit also affected signal and noise. When the plasmid harboring three copies of the reporter unit was used, the signal-to-noise ratio doubled and the detection limit decreased from 20 to 7.5 microg L(-1) As(III), compared to the use of the plasmid harboring one copy of the ars promoter/operator-arsR-gfp. Thus, segregation of arsR from the ars promoter/operator-gfp using two plasmids is effective in regulating the signal-to-noise ratio and the detection limit with the different functions.

Carotenoid production in Bacillus subtilis achieved by metabolic engineering.

The carotenoid synthetic genes, crtM and crtN, derived from Staphylococcus aureus, were introduced into B. subtilis, resulting in yellow pigmentation. Absorption maxima of pigments and MALDI-TOF mass spectrometry demonstrated that the pigmented strain accumulated two C(30) carotenoids, 4,4'-diapolycopene and 4,4'-diaponeurosporene. A survival test using H(2)O(2) revealed that the pigmented strain was more resistant to oxidative stress than the strain harboring an empty-vector. These findings indicate that B. subtilis can produce carotenoids, and the strain accumulating the carotenoids, CarotenoBacillus, will become a basal host for production of C(30) carotenoids and evaluation of their antioxidative effects.

Novel carotenoid-based biosensor for simple visual detection of arsenite: characterization and preliminary evaluation for environmental application.

A novel whole-cell arsenite biosensor was developed using the photosynthetic bacterium Rhodopseudomonas palustris no. 7 and characterized. A sensor plasmid containing the operator-promoter region of the ars operon and arsR gene from Escherichia coli and the crtI gene from R. palustris no. 7 was introduced into a blue-green mutant with crtI deleted, R. palustris no. 711. The biosensor changed color in response to arsenite, and the change was obvious to the naked eye after 24 h without further manipulation. Real-time reverse transcription-PCR showed that the crtI mRNA was induced 3-fold at 3 h and 2.5-fold at 6 h after addition of 50 microg/liter arsenite compared with the no-arsenite control, and consistent with this, the relative levels of lycopene and rhodopin also increased compared with the control. Colorimetric analysis of the bacteria showed that the hue angle had clearly shifted from green-yellow toward red in an arsenic dose-dependent manner at 24 h after arsenite addition. This obvious shift occurred irrespective of the culture conditions before arsenite was added, indicating that the color change of the biosensor is stable in water samples containing various concentrations of dissolved oxygen. Finally, assays using samples prepared in various types of mineral water indicated that this biosensor could be used to screen groundwater samples for the presence of arsenite in a variety of locations, even where electricity is not available.

Functional substitution of the transient membrane-anchor domain in Escherichia coli FtsY with an N-terminal hydrophobic segment of Streptomyces lividans FtsY.

FtsY is a signal recognition particle receptor in Escherichia coli that mediates the targeting of integral membrane proteins to translocons by interacting with both signal recognition particle (SRP)-nascent polypeptide-ribosome complexes and the cytoplasmic membrane. Genes encoding the N-terminal segments of Streptomyces lividans FtsY were fused to a gene encoding the E. coli FtsY NG domain (truncated versions of FtsY lacking the transient membrane-anchor domain at the N-terminus), introduced into a conditional ftsY-deletion mutant of E. coli, and expressed in trans to produce chimeric FtsY proteins. Under FtsY-depleted conditions, strains producing chimeric proteins including 34 N-terminal hydrophobic residues grew whereas strains producing chimeric proteins without these 34 residues did not. A strain producing the chimeric protein comprising the 34 residues and NG domain processed beta-lactamase, suggesting that the SRP-dependent membrane integration of leader peptidase was restored in this strain. These results suggest that the N-terminal hydrophobic segment of FtsY in this Gram-positive bacterium is responsible for its interaction with the cytoplasmic membrane.

Mutational study on alphaGln90 of Fe-type nitrile hydratase from Rhodococcus sp. N771.

Nitrile hydratase (NHase) from Rhodococcus sp. N771 is a non-heme iron enzyme having post-translationally modified cysteine ligands, alphaCys112-SO2H and alphaCys114-SOH. We replaced alphaGln90, which is conserved in all known NHases and involved in the hydrogen-bond network around the catalytic center, with glutamic acid or asparagine. The kcat of alphaQ90E and alphaQ90N mutants decreased to 24% and 5% that of wild type respectively, but the effect of mutations on Km was not very significant. In both mutants, the alphaCys114-SOH modification appeared to be responsible for the catalysis as in native NHase. We crystallized the nitrosylated alphaQ90N mutant and determined its structure at a resolution of 1.43 A. The structure was basically identical to that of native nitrosylated NHase except for the mutated site and its vicinity. The structural difference between native and alphaQ90N mutant NHases suggested the importance of the hydrogen bond networks between alphaGln90 and the iron center for the catalytic activity.

Simultaneous control of turbidity and dilution rate through adjustment of medium composition in semi-continuous Chlamydomonas cultures.

For production of starch in algal cultures, a growth rate limited by a nutrient is an important factor. Under phototrophic conditions, turbidity must be also paid attention, as the shading effect may affect its productivity. Semi-continuous cultivation methods, which enable control of turbidity and dilution rate (D) at the same time, have been developed for evaluation of those factors on starch production in Chlamydomonas sp. A specific feature of the methods is in a process of alternately feeding medium adjusted at two different nitrogen (N) concentrations. In the turbidostat-based method, a turbidostat culture was operated repeating three steps of determining D within a preset interval, alternating media by comparing the D with a preset value, and adjusting D in the next interval by feeding the selected medium. In the chemostat-based method, turbidity of a chemostat culture was controlled by repeating two steps of alternating media by comparing transmitted photon flux intensity (I) with a preset value and adjusting I by feeding the selected medium. D controlled by the turbidostat-based method reached quickly a preset value as low as 0.010/h, and then it was dispersed around but above the preset value. On the other hand, mean N concentrations of fed media formed a plateau. In the chemostat-based method, I was well controlled to a preset value while the mean N concentrations were a bit fluctuated. Starch concentration varied from 0.052 to 0.41 g/L with turbidity and D defined by these methods.

Colorimetric dimethyl sulfide sensor using Rhodovulum sulfidophilum cells based on intrinsic pigment conversion by CrtA.

A colorimetric whole-cell sensor for dimethyl sulfide (DMS) was constructed based on the in vivo conversion of intrinsic pigments in response to the analyte. In a marine bacterium, Rhodovulum sulfidophilum, carotenoids are synthesized via the spheroidene pathway. In this pathway, demethylspheroidene, a yellow carotenoid, is converted to spheroidene under catalysis of O-methyltransferase. Spheroidene monooxygenase (CrtA) catalyzes the terminal step of the pathway and converts spheroidene to spheroidenone, a red carotenoid. Here, the CrtA gene in R. sulfidophilum was removed and then reintroduced downstream of the DMS dehydrogenase gene promoter. Using this whole-cell sensor, 3 muM DMS or dimethyl sulfoxide can be detected without adding any color-forming reagent. The ratio of the red spheroidenone to total carotenoids increased, as the DMS concentration was raised to 0.3 mM. Comparison of the signal to the background color indicated a shift in the color coordinate from a yellow to a red hue. An intense signal was obtained with 1-day incubation at a high cell density when sensor cells at the exponential growth phase were used. These results show that the genetically engineered R. sulfidophilum cells can be used to monitor the quality of marine aquacultural environments by the naked eye.

Unusual accumulation of demethylspheroidene in anaerobic-phototrophic growth of crtA-deleted mutants of Rhodovulum sulfidophilum.

Rhodovulum sulfidophilum produces carotenoids in the spheroidene pathway. Spheroidene monooxygenase, CrtA, catalyzes the conversion of spheroidene to spheroidenone. crtA-deleted mutants of R. sulfidophilum did not produce spheroidenone and demethylspheroidenone. In these mutants, the ratio of demethylspheroidene to spheroidene increased with exposure to light. One mutant exhibiting a spheroidene-predominant phenotype did not grow under anaerobic-light conditions and was devoid of bacteriochlorophyll a, even under semiaerobic-light conditions There was no difference in the growth of the mutants under aerobic-dark conditions. These data suggest that demethylspheroidene is important for photosynthesis in R. sulfidophilum.

Expression profiles of polyhydroxyalkanoate synthesis-related genes in Paracoccus denitrificans.

A facultative methylotrophic bacterium, Paracoccus denitrificans can synthesize polyhydroxyalkanoate acids (PHA) from various alcohols. Recently, six genes, phaA, B, C, P, R, and Z, related to PHA synthesis have been cloned and characterized. PHA synthesis and the expression of phaA, B, C, P, R, and Z in P. denitrificans were examined at the transcriptional and translational levels under both nitrogen-sufficient and nitrogen-deficient conditions. The results showed that PHA synthesis is not regulated at the mRNA or protein level in phaA, B, and C. We also observed the condensation of acetyl-coenzyme A (acetyl-CoA) in the cells by high-performance liquid chromatography (HPLC). The results suggest that the amount of acetyl-CoA would regulate PHA synthesis. Finally, we discuss a possible regulation mechanism for PHA synthesis in P. denitrificans.

Purification and properties of D-(-)-3-hydroxybutyrate oligomer hydrolase of Paracoccus denitrificans.

D-(-)-3-Hydroxybutyrate (3HB) oligomer hydrolase was purified from Paracoccus denitrificans. The enzyme was a monomeric protein with an approximate molecular mass of 31 kDa. The isoelectric point of the enzyme was 5.2. Optimum temperature and pH were 35-40 degrees C and 8.0, respectively. The enzyme activity was not affected by sulfhydryl reagents but strongly inhibited by serine proteinase inhibitors. Both 3HB trimer and 3HB dimer were hydrolyzed by the enzyme, indicating that the enzyme is not 3HB dimer hydrolase but 3HB oligomer hydrolase. para-Nitrophenyl esters of short-chain fatty acids were also hydrolyzed by the enzyme. 3HB dimer was hydrolyzed somewhat faster than 3HB trimer. The level of the enzyme activity was almost constant, irrespective of carbon sources for the bacterial growth and of the cultivation conditions.