Biotests for mineral waters with natural and recombinant luminescent microorganisms

We have developed methods of biotesting mineral waters involving use of natural or recombinant luminescent strains with elimination of the effect of salt concentration and pH. To overcome the adverse effect of high salt concentrations, disguising the action of chemical pollutants, a special method of mineral water sample preparation is proposed. In this method, the marine luminescent bacterium Photobacterium phosphoreum (Microbiosensor B17 677f) is used as a test object. Samples to be analyzed are supplemented with NaCl depending on their natural salt concentration to adjust it to 3 g/l. Another approach, more universal and efficient, involves pH adjustment in the samples to 7.5. This value is suitable for application of both Microbiosensor B17 677f and the recombinant Escherichia coli strain harboring the cloned lux operon of P. leiognathi (Ecolum 9). It has been shown that this treatment, retaining the natural luminescence level of the bacterial biosensors, allows bioluminescent detection of exogenous pollutants added to the samples, including benzene and Cr(VI).     

Effect of salts on luminescence of natural and recombinant luminescent bacterial biosensors

Effect of cations K+, Na+, Mg2+, and Ca2+ and anions SO4(2-), HCO3(-), and CO3(2-) on the luminescence intensity of the marine luminescent bacterium Photobacterium phorphoreum (Microbiosensor B-17 677f) and the recombinant strain Escherichia coli with cloned lux operon of P. leiognathi (Ekolyum-9). It is found that small concentrations of chlorides and sulfates of the cations studied had a concentration-dependent stimulatory effect on bacterial bioluminescence; as the concentration of agents increased, activation was succeeded by quenching. The strength of the inhibitory effect, which is characterized by EC50, decreased in the series Ca2+ > Na+ > Mg2+ > K+. Carbonates and hydrocarbonates had a pronounced inhibitory effect on the bioluminescence intensity, determined by an increase in pH. We showed that some types of highly mineralized water with a high hydrocarbonate content have a marked inhibitory effect on the luminescence intensity of microbial luminescent biosensors, mimicking the effect of chemical pollutants.     

Effect of salts on luminescence of natural and recombinant luminescent bacterial biosensors

Effect of cations K⁺, Na⁺, Mg²⁺, and Ca²⁺ and anions Cl^?, SO ₄ ^2? , HCO ₃ ^? , and CO ₃ ^2? on the luminescence intensity of the marine luminescent bacterium Photobacterium phorphoreum (Microbiosensor B-17 677f) and the recombinant strain Escherichia coli with cloned lux operon of P. leiognathi (Ecolum-9). It is found that small concentrations of chlorides and sulfates of the cations studied had a concentration-dependent stimulatory effect on bacterial bioluminescence; as the concentration of agents increased, activation was succeeded by quenching. The strength of the inhibitory effect, which is characterized by EC₅₀, decreased in the series Ca²⁺ > Na⁺ > Mg²⁺ > K⁺. Carbonates and hydrocarbonates had a pronounced inhibitory effect on the bioluminescence intensity, determined by an increase in pH. We showed that some types of highly mineralized water with a high hydrocarbonate content have a marked inhibitory effect on the luminescence intensity of microbial luminescent biosensors, mimicking the effect of chemical pollutants.     

IL-17A Promotes Pulmonary B-1a Cell Differentiation via Induction of Blimp-1 Expression during Influenza Virus Infection

B-1 cells play a critical role in early protection during influenza infections by producing natural IgM antibodies. However, the underlying mechanisms involved in regulating this process are largely unknown. Here we found that during influenza infection pleural cavity B-1a cells rapidly infiltrated lungs, where they underwent plasmacytic differentiation with enhanced IgM production. This process was promoted by IL-17A signaling via induction of Blimp-1 expression and NF-κB activation in B-1a cells. Deficiency of IL-17A led to severely impaired B-1a-derived antibody production in the respiratory tract, resulting in a deficiency in viral clearance. Transfer of B-1a-derived natural antibodies rescued Il17a ^-/- mice from otherwise lethal infections. Together, we identify a critical function of IL-17A in promoting the plasmacytic differentiation of B-1a cells. Our findings provide new insights into the mechanisms underlying the regulation of pulmonary B-1a cell response against influenza infection.     

Insights from the draft genome of Paenibacillus lentimorbus NRRL B-30488, a promising plant growth promoting bacterium

Paenibacillus lentimorbus NRRL B-30488, a plant growth-promoting bacterium was isolated from Sahiwal cow's milk. The strain shows antagonism against phytopathogens, Fusarium oxysporum f. sp. ciceri and Alternaria solani. Its genome contains gene clusters involved in nonribosomal synthesis of secondary metabolites involved in antimicrobial activities. The genome sequence of P. lentimorbus NRRL B-30488 provides the genetic basis for application of this bacterial strain in plant growth promotion, plant protection and degradation of organic pollutants.     

Conditions that influence bacterial luminescence in the presence of blood serum

Conditions that influence the luminescence of natural and recombinant luminescent bacteria in the presence of blood serum were studied. In general, blood serum quenched the luminescence of the marine Photobacterium phosphoreum and the recombinant Escherichia coli strains harboring the luminescent system genes of Photobacterium leiognathi, but enhanced the luminescence of the soil bacterium Photorhabdus luminescens Zm1 and the recombinant E. coli strain harboring the lux operon of P. luminescens Zm1. The quenching effect of blood serum increased with its concentration and the time and temperature of incubation. The components of blood serum that determine the degree and specificity of its action on bacterial luminescence were identified.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 191–197.Original Russian Text Copyright © 2005 by Deryabin, Polyakov.     

The proteolytic processing of amelogenin by enamel matrix metalloproteinase (MMP-20) is controlled by mineral ions

Background

Enamel synthesis is a highly dynamic process characterized by simultaneity of matrix secretion, assembly and processing during apatite mineralization. MMP-20 is the first protease to hydrolyze amelogenin, resulting in specific cleavage products that self-assemble into nanostructures at specific mineral compositions and pH. In this investigation, enzyme kinetics of MMP-20 proteolysis of recombinant full-length human amelogenin (rH174) under different mineral compositions is elucidated.

Methods

Recombinant amelogenin was cleaved by MMP-20 under various physicochemical conditions and the products were analyzed by SDS-PAGE and MALDI-TOF MS.

Results

It was observed that mineral ions largely affect cleavage pattern, and enzyme kinetics of rH174 hydrolysis. Out of the five selected mineral ion compositions, MMP-20 was most efficient at high calcium concentration, whereas it was slowest at high phosphate, and at high calcium and phosphate concentrations. In most of the compositions, N- and C-termini were cleaved rapidly at several places but the central region of amelogenin was protected up to some extent in solutions with high calcium and phosphate contents.

Conclusion

These in vitro studies showed that the chemistry of the protein solutions can significantly alter the processing of amelogenin by MMP-20, which may have significant effects in vivo matrix assembly and subsequent calcium phosphate mineralization.

General significance

This study elaborates the possibilities of the processing of the organic matrix into mineralized tissue during enamel development.     

MV-mimicking micelles loaded with PEG-serine-ACP nanoparticles to achieve biomimetic intra/extra fibrillar mineralization of collagen in vitro

Since their discovery, matrix vesicles (MVs) containing minerals have received considerable attention for their role in the mineralization of bone, dentin and calcified cartilage. Additionally, MVs' association with collagen fibrils, which serve as the scaffold for calcification in the organic matrix, has been repeatedly highlighted. The primary purpose of the present study was to establish a MVs–mimicking model (PEG-S-ACP/micelle) in vitro for studying the exact mechanism of MVs-mediated extra/intra fibrillar mineralization of collagen in vivo. In this study, high-concentration serine was used to stabilize the amorphous calcium phosphate (S-ACP), which was subsequently mixed with polyethylene glycol (PEG) to form PEG-S-ACP nanoparticles. The nanoparticles were loaded in the polysorbate 80 micelle through a micelle self-assembly process in an aqueous environment. This MVs–mimicking model is referred to as the PEG-S-ACP/micelle model. By adjusting the pH and surface tension of the PEG-S-ACP/micelle, two forms of minerals (crystalline mineral nodules and ACP nanoparticles) were released to achieve the extrafibrillar and intrafibrillar mineralization, respectively. This in vitro mineralization process reproduced the mineral nodules mediating in vivo extrafibrillar mineralization and provided key insights into a possible mechanism of biomineralization by which in vivo intrafibrillar mineralization could be induced by ACP nanoparticles released from MVs. Also, the PEG-S-ACP/micelle model provides a promising methodology to prepare mineralized collagen scaffolds for repairing bone defects in bone tissue engineering.     

Identification of water-conditioned Pseudomonas aeruginosa by Raman microspectroscopy on a single cell level

The identification of Pseudomonas aeruginosa from samples of bottled natural mineral water by the analysis of subcultures is time consuming and other species of the authentic Pseudomonas group can be a problem. Therefore, this study aimed to investigate the influence of different aquatic environmental conditions (pH, mineral content) and growth phases on the cultivation-free differentiation between water-conditioned Pseudomonas spp. by applying Raman microspectroscopy. The final dataset was comprised of over 7500 single-cell Raman spectra, including the species Pseudomonas aeruginosa, P. fluorescens and P. putida, in order to prove the feasibility of the introduced approach. The collection of spectra was standardized by automated measurements of viable stained bacterial cells. The discrimination was influenced by the growth phase at the beginning of the water adaptation period and by the type of mineral water. Different combinations of the parameters were tested and they resulted in accuracies of up to 85% for the identification of P. aeruginosa from independent samples by applying chemometric analysis.     

The mechanism by which tetracycline hydrochloride inhibits mineralization in vitro

1.

1. The inhibitory action of tetracycline hydrochloride on mineralization was demonstrated in vitro. Low concentrations of the drug interfered with teh precipitation of apatites from supersaturated mineralization buffers at 37° and pH 7.4. In the presence of 20 μM tetracycline, the ionic produce of calcium and phosphate required for spontaneous precipitation rose from 2.5 to 4.0 (mM)². The amount of mineral in the precipitate was not altered, which suggests that tetracycline affects the primary nucleation of mineralization.     

Biodegradation of Aromatic Hydrocarbons in an Extremely Acidic Environment

The potential for biodegradation of aromatic hydrocarbons was evaluated in soil samples recovered along gradients of both contaminant levels and pH values existing downstream of a long-term coal pile storage basin. pH values for areas greatly impacted by runoff from the storage basin were 2.0. Even at such a reduced pH, the indigenous microbial community was metabolically active, showing the ability to oxidize more than 40% of the parent hydrocarbons, naphthalene and toluene, to carbon dioxide and water. Treatment of the soil samples with cycloheximide inhibited mineralization of the aromatic substrates. DNA hybridization analysis indicated that whole-community nucleic acids recovered from these samples did not hybridize with genes, such as nahA, nahG, nahH, todC1C2, and tomA, that encode common enzymes from neutrophilic bacteria. Since these data suggested that the degradation of aromatic compounds may involve a microbial consortium instead of individual acidophilic bacteria, experiments using microorganisms isolated from these samples were initiated. While no defined mixed cultures were able to evolve ¹⁴CO₂ from labeled substrates in these mineralization experiments, an undefined mixed culture including a fungus, a yeast, and several bacteria successfully metabolized approximately 27% of supplied naphthalene after 1 week. This study shows that biodegradation of aromatic hydrocarbons can occur in environments with extremely low pH values.     

Stepwise synthesis of methyl 4-O-[3-O-(β-d-xylopyranosyl)-β-d-xylopyranosyl]-β-d-xylopyranoside

The general properties and specificity of a dextran α-(1→2)-debranching enzyme from Flavobacterium have been examined in order to apply this enzyme to the structural analysis of highly branched dextrans. The optimum pH range and temperature were pH 5.5–6.5, and 45°, respectively. The enzyme was stable up to 40° on heating for 10 min, and over a pH range of 6.5–9.0 on incubation at 4° for 24 h. The effects of various metal ions and chemical reagents have also been examined. The debranching enzyme has a strict specificity for the (1→2)-α-d-glucosidic linkage at branch points of dextrans and related branched oligosaccharides, and produces d-glucose as the only reducing sugar. The degree of hydrolysis of the dextrans by this enzyme and the K_m value (mg/mL) were as follows: B-1298 soluble, 25.2%, 0.21; B-1299 soluble, 31.5%, 0.27; and B-1397, 11.8%, 0.91. The debranching enzyme thus has a novel type of specificity as a dextranhydrolase. We have termed this enzyme as dextran α-(1→2)-debranching enzyme, and its systematic name is also discussed.     

The influence of western hemlock and western redcedar on microbial numbers,nitrogen mineralization,and nitrification

Summary Microbial numbers in the forest floor and mineral soil (Al horizon) under large individual western hemlock (Tsuga heterophylla) and western redcedar (Thuja plicata) trees were compared. The lower pH and base saturation of hemlock samples was associated with higher fungal spore counts while cedar samples had higher total microbial counts and populations of ammonium oxidizing bacteria. Nitrogen mineralization rates were greater in laboratory incubations of hemlock soil but nitrification was only observed in incubations of cedar soil. These differences in nitrogen mineralization and nitrification are aspects of species-specific nutrient cycling regimes.     

Vitamin B-6 requirement for irreversible inactivation of rat liver tyrosine aminotransferase.

In vitro inactivation of tyrosine aminotransferase at pH 7.0 did not occur in liver homogenates prepared from vitamin B-6-deficient rats, although it was previously demonstrated that the enzyme was inactivated in liver homogenates from vitamin B-6-adequate rats (R. D. Reynolds and S. D. Thompson, 1974, Arch. Biochem. Biophys.164, 43–51). Addition of 2 mm pyridoxine or pyridoxal-P to the incubated homogenate did not restore the inactivation, but injection of 1 mg of pyridoxine to deficient rats restored full inactivating activity by 12 h. All forms of vitamin B-6 injected restored inactivating activity in vitro. This effect appears to be specific for vitamin B-6, since no restoration of in vitro inactivation of tyrosine aminotransferase was observed following injection of riboflavin, thiamin, niacin, or folic acid. The restoration of inactivating activity in vitro following injection of pyridoxine was not inhibited by repeated injections of puromycin or cycloheximide. Apparently, in vivo protein synthesis is not required for the restoration of the in vitro inactivating activity. However, in vivo inactivation was similar in the vitamin B-6-adequate and -deficient rats. Inactivating activity is present in homogenates of liver and kidney, but not of abdominal muscle, small intestine, heart, testes, whole blood, or erythrocyte ghosts, and is found only in the plasma membrane fraction of liver. Similar to liver, the activity in the kidney homogenate requires the presence of l-cysteine and depends upon the vitamin B-6 status of the animal. Rapid inactivation in the liver occurs between pH 6.75 and 7.75 (final pH), with minimal inactivation above or below this range. No inhibition of inactivation was observed with homogenates incubated in the presence of several protease inhibitors.     

Accelerated Growth Plate Mineralization and Foreshortened Proximal Limb Bones in Fetuin-A Knockout Mice

The plasma protein fetuin-A/alpha2-HS-glycoprotein (genetic symbol Ahsg) is a systemic inhibitor of extraskeletal mineralization, which is best underscored by the excessive mineral deposition found in various tissues of fetuin-A deficient mice on the calcification-prone genetic background DBA/2. Fetuin-A is known to accumulate in the bone matrix thus an effect of fetuin-A on skeletal mineralization is expected. We examined the bones of fetuin-A deficient mice maintained on a C57BL/6 genetic background to avoid bone disease secondary to renal calcification. Here, we show that fetuin-A deficient mice display normal trabecular bone mass in the spine, but increased cortical thickness in the femur. Bone material properties, as well as mineral and collagen characteristics of cortical bone were unaffected by the absence of fetuin-A. In contrast, the long bones especially proximal limb bones were severely stunted in fetuin-A deficient mice compared to wildtype littermates, resulting in increased biomechanical stability of fetuin-A deficient femora in three-point-bending tests. Elevated backscattered electron signal intensities reflected an increased mineral content in the growth plates of fetuin-A deficient long bones, corroborating its physiological role as an inhibitor of excessive mineralization in the growth plate cartilage matrix - a site of vigorous physiological mineralization. We show that in the case of fetuin-A deficiency, active mineralization inhibition is a necessity for proper long bone growth.     

The effect of the pH level on the communities of anoxygenic phototrophic bacteria of soda lakes of the southeastern Transbaikal Region

The effect of pH on the structure of the communities of anoxygenic phototrophic bacteria (APB) was studied under laboratory conditions. Samples of natural APB communities were inoculated into media that differed in pH values, which were 7, 9.5, or 10.5. The structure of the APB communities in the obtained enrichment cultures at all pH values depended also on the mineralization levels of the media, which were the same as in the lakes from which samples were taken. The same dependence of the community structure on salinity was observed as in the case of the natural communities that had been described previously. APB were most diverse in the enrichment cultures grown at pH 9.5. The shift of the pH to either neutral or extremely alkaline values restricted the species diversity within the APB community, resulting in marked predominance of the most adapted forms. It was shown that the status of Ectothiorhodospira species within the community could serve not only as an indicator of salinity but also as an indicator of pH in soda lakes with a water mineralization of higher than 5 g/l. The statuses of various APB groups in the community as dependent on pH and salinity are discussed, as well as possible changes in these statuses due to changes in the water level and other environmental parameters in the studied lakes.     

Effect of Substrate Concentration and Organic and Inorganic Compounds on the Occurrence and Rate of Mineralization and Cometabolism

Isopropyl N-phenylcarbamate (IPC) at 400 pg and 1 μg/ml was mineralized in samples of sewage, but only the lower concentration was mineralized in lake water samples in a 50-day period. IPC at 1 μg/ml disappeared from lake water, but it was converted to organic products. Mineralization of IPC at 400 pg/ml in lake water was enhanced by additions of inorganic nutrients or a mixture of nonchlorinated water pollutants but not by yeast extract or mixtures containing aromatic compounds or excretions of primary producers. The mineralization of 200 pg of 2,4-dichlorophenoxyacetate per ml of lake water was not affected by additions of low levels of yeast extract or compounds excreted by primary producers but was enhanced by low concentrations of mixtures of water pollutants. It is suggested that some chemicals that are found to be converted only to organic products, presumably by cometabolism, in tests using the concentrations commonly employed in laboratory evaluations may be mineralized at the lower concentrations prevailing in natural waters.     

An improved synthesis of 1,6-anhydro-2,4-dideoxy-β-D-threo-hexopyranose

Dextran fractions from NRRL strains Leuconostoc mesenteroides B-1299 and B-1399, and the native, structurally homogeneous dextrans from L. mesenteroides. B-640, B-1396, B-1422, and B-1424, were examined by ¹³C-n.m.r. spectroscopy at 34 and at 90°, and by g.l.c.-m.s. The ¹³C-n.m.r. data indicate that the dextrans of this series branch exclusively through α-d-(1→2)-linkages, and differ from one another only in degree of linearity. Diagnostic, ¹³C-n.m.r resonances, correlating with 2,6-di-O-substituted α-d-glucosyl residues at branch points, have chemical shifts that are independent of the degree of linearity of the dextran. The intensities of these diagnostic resonances from branching residues, compared to the resonances associated with linear dextran (low degree of branching), are generally proportional to the degree of branching established by methylation-fragmentation analysis. The validity of assignment of the diagnostic, ¹³C-n. m.r. resonances is substantiated by a critical review of methods previously used to provide structural information on dextrans having α-d-(1→2)-linkages, and by evaluation of the corresponding results on the basis of the ultimate standard-methylation structural analysis.     

Evaluation of High Resolution Melting for MTHFR C677T Genotyping in Congenital Heart Disease

Background

High resolution melting (HRM) is a simple, flexible and low-cost mutation screening technique. The methylenetetrahydrofolate reductase (MTHFR) gene encoding a critical enzyme, potentially affects susceptibility to some congenital defects like congenital heart disease (CHD). We evaluate the performance of HRM for genotyping of the MTHFR gene C677T locus in CHD cases and healthy controls of Chinese Han population.

Methods

A total of 315 blood samples from 147 CHD patients (male72, female 75) and 168 healthy controls (male 92, female 76) were enrolled in the study. HRM was utilized to genotype MTHFR C677T locus of all the samples. The results were compared to that of PCR-RFLP and Sanger sequencing. The association of the MTHFR C677T genotypes and the risk of CHD was analyzed using odds ratio with their 95% confidence interval (CIs) from unconditional logistic regression.

Results

All the samples were successfully genotyped by HRM within 1 hour and 30 minutes while at least 6 hours were needed for PCR-RFLP and sequencing. The genotypes of MTHFR C677T CC, CT, and TT were 9.52%, 49.66%, and 40.82% in CHD group but 29.17%, 50% and 20.83% in control group, which were identical using both methods of HRM and PCR-RFLP, demonstrating the sensitivity and specificity of HRM were all 100%.

Conclusion

MTHFR C677T is a potential risk factor for CHD in our local residents of Shandong province in China. HRM is a fast, sensitive, specific and reliable method for clinical application of genotyping.     

Isolation and characterization of a thermotolerant ene reductase from Geobacillus sp. 30 and its heterologous expression in Rhodococcus opacus

Rhodococcus opacus B-4 cells are adhesive to and even dispersible in water-immiscible hydrocarbons owing to their highly lipophilic nature. In this study, we focused on the high operational stability of thermophilic enzymes and applied them to a biocatalytic conversion in an organic reaction medium using R. opacus B-4 as a lipophilic capsule of enzymes to deliver them into the organic medium. A novel thermo- and organic-solvent-tolerant ene reductase, which can catalyze the enantioselective reduction of ketoisophorone to (6R)-levodione, was isolated from Geobacillus sp. 30, and the gene encoding the enzyme was heterologously expressed in R. opacus B-4. Another thermophilic enzyme which catalyzes NAD⁺-dependent dehydrogenation of cyclohexanol was identified from the gene-expression library of Thermus thermophilus and the gene was coexpressed in R. opacus B-4 for cofactor regeneration. While the recombinant cells were not viable in the mixture due to high reaction temperature, 634 mM of (6R)-levodione could be produced with an enantiopurity of 89.2 % ee by directly mixing the wet cells of the recombinant R. opacus with a mixture of ketoisophorone and cyclohexanol at 50 °C. The conversion rate observed with the heat-killed recombinant cells was considerably higher than that obtained with a cell-free enzyme solution, demonstrating that the accessibility between the substrates and enzymes could be improved by employing R. opacus cells as a lipophilic enzyme capsule. These results imply that a combination of thermophilic enzymes and lipophilic cells can be a promising approach for the biocatalytic production of water-insoluble chemicals.