A thermophilic process for catechol production from phenol

Summary A phenol tolerant thermophile,Bacillus stearothermophilus BR219, catabolizes phenol via a plasmid encoded catecholmeta pathway. Direct and selective inhibition of catechol 2,3-dioxygenase encoded in this pathway by tetracycline results in accumulation of the specialty chemical catechol. Optimal conditions for catechol production are presented.     

Conjugal transfer of recombinant transposon Tn916 from Escherichia coli to Bacillus stearothermophilus.

A gene for thermostable amylase has been inserted at the BstXI site of Tn916. Mating experiments demonstrated that unlike Tn916, the recombinant transposon, designated Tn916A, could transfer from Escherichia coli to Bacillus stearothermophilus BR219 in broth matings, resulting in chromosomal integration of the transposon and expression of the amylase at significant levels.     

Transfer of Transposon Tn916 from Bacillus subtilis into a Natural Soil Population

A Bacillus subtilis transconjugant with a Tn916 chromosomal insert was obtained through mating with Escherichia coli carrying the transposon as a plasmid insert. Actinomycetes were identified as frequent transposon recipients following the introduction of the B. subtilis transconjugant into a soil microcosm.     

Cloning and expression of the nitrile hydratase and amidase genes from Bacillus sp. BR449 into Escherichia coli

A moderate thermophile, Bacillus sp. BR449 was previously shown to exhibit a high level of nitrile hydratase (NHase) activity when growing on high levels of acrylonitrile at 55 degrees C. In this report, we describe the cloning of a 6.1 kb SalI DNA fragment encoding the NHase gene cluster of BR449 into Escherichia coli. Nucleotide sequencing revealed six ORFs encoding (in order), two unidentified putative proteins, amidase, NHase beta- and alpha-subunits and a small putative protein of 101 amino acids designated P12K. Spacings and orientation of the coding regions as well as their gene expression in E. coli suggest that the beta-subunit, alpha-subunit, and P12K genes are co-transcribed. Analysis of deduced amino acid sequences indicate that the amidase (348 aa, MW 38.6 kDa) belongs to the nitrilase-related aliphatic amidase family, and that the NHase beta- (229 aa, MW 26.5 kDa) and alpha- (214 aa, MW 24.5 kDa) subunits comprise a cobalt-containing member of the NHase family, which includes Rhodococcus rhodochrous J1 and Pseudomonas putida 5B NHases. The amidase/NHase gene cluster differs both in arrangement and composition from those described for other NHase-producing strains. When expressed in Escherichia coli DH5alpha, the subcloned NHase genes produced significant levels of active NHase enzyme when cobalt ion was added either to the culture medium or cell extracts. Presence of the P12K gene and addition of amide compounds as inducers were not required for this expression.     

Gentisate 1,2-dioxygenase from Haloferax sp. D1227

Gentisate 1,2-dioxygenase from the extreme halophile Haloferax sp. D1227 (Hf. D1227) was purified using a three-step procedure. The enzyme was found to be a homotetramer of 42 000 ± 1000 Da subunits, with a native molecular weight of 174 000 ± 6000 Da. The optimal salt concentration, temperature, and pH for enzyme activity were 2 M KCl or NaCl, 45°C, and pH 7.2, respectively. The gene encoding Hf. D1227 gentisate 1,2-dioxygenase was cloned, sequenced, and expressed in Haloferax volcanii. The deduced amino acid sequence exhibited a 9.2% excess acidic over basic amino acids typical of halophilic enzymes. Four novel histidine clusters and a possible extradiol dioxygenase fingerprint region were identified. Received: November 19, 1997 / Accepted: May 12, 1998     

Photoinactivation of Candida albicans by Its Own Endogenous Porphyrins

The possibility of photoeradicating the prokaryotic microorganism Candida albicans by enhancing its endogenous porphyrin production and accumulation was investigated in this study. Induction of porphyrin synthesis was performed by the addition of δ-aminolevulinic acid (ALA), or its hydrophobic derivative ALA methyl ester (m-ALA). Photoinactivation of C. albicans was performed under blue light (407–420 nm) illumination. A decrease in viability of about 1.6 or 2.1 orders of magnitudes was obtained with a light dose of 36 J/cm² for an initial concentration of 100-mg/ml ALA or m-ALA, respectively. Endogenous porphyrins extracted from the cells showed that cultures incubated with m-ALA accumulated a relatively higher amount of endogenous porphyrins than ALA, indicating better transport through the yeast cell barriers. When a combination of miconazole and ketoconazole (antifungal agents) is given at a sub-inhibitory concentration (0.5 μg/ml each) with an inducer, a 2.1 or 3.2 orders of magnitude decrease in viability is caused with ALA or with m-ALA, respectively, upon illumination. Fluorescence intensities of the accumulated porphyrins as demonstrated by FACS indicate that the combination of the two azole drugs and an inducer cause a relatively high amount of endogenous porphyrins. Although the additive action of both azole drugs allow better penetration of the inducer, especially m-ALA photoeradication remained limited because of an acidic pH generated in the presence of the inducer. The acidic pH is probably the cause for the inefficiency of the photodynamic treatment. More hydrophobic inducers than m-ALA and less acidic must be investigated to improve the photodynamic treatment by endogenous-induced porphyrins.     

Loop-mediated isothermal amplification (LAMP) assays for detection of Theileria parva infections targeting the PIM and p150 genes

We have developed two loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria parva, the causative agent of East Coast fever (ECF), an economically important cattle disease in eastern, central and southern Africa. These assays target the polymorphic immunodominant molecule (PIM) and p150 LAMP genes. The primer set for each gene target consists of six primers, and each set recognises eight distinct regions on the target gene to give highly specific detection of T. parva. The detection limit of each primer set is 1 fg, which is equivalent to one copy of the PIM and p150 T. parva genes. These PIM and p150 LAMP primer sets amplify DNA of T. parva isolates from cattle and buffalo from different countries including Kenya, South Africa, Tanzania, Rwanda, Uganda and Burundi, indicating their ability to detect T. parva from different countries. With the advantages of simplicity, rapidity and cost effectiveness, these LAMP assays are good candidates for molecular epidemiology studies and for monitoring control programs in ECF-endemic, resource poor countries.     

Studies of the birefringence and birefringence dispersion of polypeptides and proteins

An apparatus has been constructed which permits the polarimetric observation of streaming solutions of macromolecules. The apparatus is a streaming birefringence device allowing the usual measurements of birefringence parallel to the cylinder axis but which in addition transmits light in the radial direction. Installation of the apparatus between the polarizer and analyzer of a Rudolph polarimeter makes possible the measurement of changes in optical rotation, dichroism and birefringence. The present work is concerned with the latter effect. The systems studied were α-helical polyglutamic acid, paramyosin, and collagen (ichthyocol). The combined measurements of radial and axial birefringence completely determines the refractive index ellipsoid of the streaming fluid. This result in turn permits the testing of the Peterlin-Stuart distribution function for streaming in a Couette device, apart from a proportionality constant. The comparison between theory and experiment is very satisfactory provided the system is reasonably homogeneous with regard to molecular length and is sufficiently dilute. On the other hand, it is concluded that the Peterlin-Stuart optical factor seriously overestimates the “form” birefringence in agreement with recent results and conclusions of Taylor and co-workers. The apparatus permits the study of the dispersion of the birefringence in the radial direction. The dispersion of collagen follows a one-term Sellmeier formula and is dominated by absorption bands in the neighborhood of 2000 A. On the other hand, the dispersion of the α-helical systems is complex and requires a multiterm Sellmeier formula. This contrast between the two kinds of polypeptidc helices is similar to results obtained with other optical techniques and is attributed to the splitting of absorption bands in the α-helix.     

The effect of α-tocopherol transfer protein gene disruption on Trypanosoma congolense infection in mice

At present 15 to 20 million people are estimated to be infected with pathogenic trypanosome parasites worldwide, mainly in developing countries. There are a number of factors that affect the severity of trypanosomiasis, including the nutritional status of the host. However, the relationship between micronutrient levels and trypanosomiasis outcome has yet to be reported in detail. Here, we demonstrate that the inhibition of α-tocopherol transfer protein, a determinant of the vitamin E concentration in host circulation, confers resistance to Trypanosoma congolense infection, evidently owing to oxidative damage to parasite DNA. These results suggest that transient inhibition of α-tocopherol transfer gene activity could possibly be exploited as a strategy for both the prevention and the treatment of trypanosomiasis.