Employment of 16S rDNA gene sequencing techniques for improved identification of difficult-to-identify bacterial veterinary pathogens

To employ 16S rDNA PCR and automated sequencing techniques to identify a collection of bacterial veterinary pathogens from avian, equine, canine and ovine sources, that have proven difficult to identify, employing conventional cultural techniques. Universal or “broad-range” eubacterial PCR was performed on a collection of 46 difficult-to-identify bacterial isolates originating from clinical veterinary specimens. 16S rDNA PCR was performed using two sets of universal primers to successfully generate a composite amplicon of 1,068 bp, which was sequenced to obtain each isolate’s identity. Sequence analysis was able to identify all isolates examined with relative ease. Where the use of molecular identification methods is justified, such as in outbreak control or bioterrorism in animal health, employment of partial 16S rDNA PCR and sequencing employing universal or “broad-range” 16S rDNA, provides a valuable and reliable method of identification of such pathogens.     

The phylogenetic relationships ofEeniella nana Smith,Batenburg-van der Vegte et Scheffers based on the partial sequences of 18S and 26S ribosomal RNAs (Candidaceae)

Summary Two strains ofEeniella nana were examined for their partial base sequences of 18S and 26S rRNAs. In the partial base sequences of 18S rRNA (prositions 1451 through 1618, 168 bases) the strains ofE. nana have five, five, four and eleven base differences with those ofDekkera bruxellensis (type species).D. anomala (andBrettanomyces anomalus),D. naardenensis andD. custersiana, respectively. In the 26S rRNA partial base sequencings (positions 1611 through 1835, 225 bases and positions 493 through 622, 130 bases) the base differences were 46, 43, 34 and 40 and the percent similarities were 53–54, 51–54, 56–57 and 51–53, respectively. The sequence data obtained are discussed phylogenetically and taxonomically, especially on retention of the generic nameEeniella.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.Significance of the coenzyme Q system in the classification of yeasts and yeast-like organisms. Part LVIII. For part LVII, see ref. [20].     

Comparison of the substrate specificity of the two bacterial desulfurization systems

Using cell-free extracts of a desulfurizing mesophile, Rhodococcus erythropolis KA2-5-1 (the Dsz system) and Escherichia coli JM109, which possesses the desulfurizing genes of a thermophile Paenibacillus sp. A11-2 (the Tds system), the reactivity of desulfurizing enzymes toward 4,6-dialkyl dibenzothiophenes (4,6-dialkyl DBTs) and 7-alkyl benzothiophenes (7-alkyl BTs) was investigated. Both systems desulfurized all the 4,6-dialkyl DBTs, except 4,6-dibutyl DBT. Although some alkylated BTs were degraded by the Dsz system, no desulfurized compounds were detected. The reactivity of the Tds system toward alkylated BTs was higher than that of DBT. In contrast to the Dsz system, the Tds system yielded desulfurized compounds from all of the alkylated BTs examined.     

Construction of hybrid photosynthetic units using peripheral and core antennae from two different species of photosynthetic bacteria: detection of the energy transfer from bacteriochlorophyll a in LH2 to bacteriochlorophyll b in LH1

Typical purple bacterial photosynthetic units consist of supra-molecular arrays of peripheral (LH2) and core (LH1-RC) antenna complexes. Recent atomic force microscopy pictures of photosynthetic units in intact membranes have revealed that the architecture of these units is variable (Scheuring et al. (2005) Biochim Bhiophys Acta 1712:109–127). In this study, we describe methods for the construction of heterologous photosynthetic units in lipid-bilayers from mixtures of purified LH2 (from Rhodopseudomonas acidophila) and LH1-RC (from Rhodopseudomonas viridis) core complexes. The architecture of these reconstituted photosynthetic units can be varied by controlling ratio of added LH2 to core complexes. The arrangement of the complexes was visualized by electron-microscopy in combination with Fourier analysis. The regular trigonal array of the core complexes seen in the native photosynthetic membrane could be regenerated in the reconstituted membranes by temperature cycling. In the presence of added LH2 complexes, this trigonal symmetry was replaced with orthorhombic symmetry. The small lattice lengths for the latter suggest that the constituent unit of the orthorhombic lattice is the LH2. Fluorescence and fluorescence-excitation spectroscopy was applied to the set of the reconstituted membranes prepared with various proportions of LH2 to core complexes. Remarkably, even though the LH2 complexes contain bacteriochlorophyll a, and the core complexes contain bacteriochlorophyll b, it was possible to demonstrate energy transfer from LH2 to the core complexes. These experiments provide a first step along the path toward investigating how changing the architecture of purple bacterial photosynthetic units affects the overall efficiency of light-harvesting.     

Peculiarities of sexual reproduction inFagus crenata forests in relation to annual production of reproductive organs

Annual production rates of reproductive organs inFagus crenata forests in the lower area of the species' range were studied using 10 litter traps in 1980–1986. The production rates of dispersed pollen were estimated by multiplying the number of fallen male inflorescences per ha per year by the mean amount of pollen per inflorescence before anthesis. Large annual fluctuations in the production rates of male and female inflorescences were recognized, whereas their annual trends were synchronized with each other. Pollen production rates were within the range 1.0–6900 (mean: 1630)×10⁹ha⁻¹ yr⁻¹, the maximum/minimum ratio attaining 7000.F. crenata was the lowest producer of pollen among seven tree species studied: the number of pollen grains equivalent to a single ovule was in the range 6.0–14×10⁴. Furthermore, the mean dry weight of a single pollen grain (3.77×10⁻⁵mg) was higher than for wind-pollinated species. Three factors seemed to cause the low seed fertility ofF. crenata. The dry-matter production rate in the best seed year reached 3252 kg ha⁻¹ yr⁻¹, of which pollen accounted for 259 kg ha⁻¹ yr⁻¹. Unproductive years with less than 10% of the maximum production occurred four times in a 7-yr period. In such years there were fewer male and female inflorescences, and more fruit dropped as a result of insect damage. Lower nut dissemination would play an important role in suppressing any increase in nut predators, and fewer flowers would be produced to avoid wastage of photosynthates in a cool-temperate climate.     

Enhanced synthesis of l - threo -3,4-dihydroxyphenylserine by high-density whole-cell biocatalyst of recombinant l -threonine aldolase from Streptomyces avelmitilis

l-threo-3,4-Dihydroxyphenylserine (DOPS) is a chiral unnatural β-hydroxy amino acid used for the treatment of Parkinson disease. We developed a continuous bioconversion system for DOPS production that uses whole-cell biocatalyst of recombinant Escherichia coli expressing l-threonine aldolase (l-TA) genes cloned from Streptomyces avelmitilis MA-4680. Maximum conversion rates were observed at 2 M glycine, 145 mM 3,4-dihydroxybenzaldehyde, 0.75% Triton-X, 5 g E. coli cells/l, pH 6.5 and 10°C. In the optimized condition, overall productivity was 8 g/l, which represents 40 times the synthesis yield possible with no optimization of conditions.     

Development of an efficient method for screening microorganisms by using symbiotic association between <Emphasis Type="Italic">Nasutitermes takasagoensis</Emphasis> and intestinal microorganisms

Screening method of microorganisms that utilized the symbiotic association between insect (Nasutitermes takasagoensis: Nt) and intestinal microorganisms was developed. The existence of desired microorganisms that grew by degrading difficult-to-degrade materials in the gut was detected using survivability of Nt as an indicator. The desired microorganisms were isolated from the survived Nt. It was thought that guts of Nt behave as continuous culture systems whereby microorganisms that cannot degrade diet components are washed out, whereas those that can degrade it are retained and concentrated in the gut. About 60% of Nt fed with phenol artificial diet (PAD) died within 7 days, while 4% of termites survived for 9 days. The structure of intestinal microorganisms of the survived Nt fed with PAD differed from the bacterial communities obtained from enrichment culture (which contained phenol) of wood-feeding Nt. Relatively high colonies (650-times) were detected in the gut of Nt fed on phenol artificial diet compared with those obtained when Nt was fed on wood. Seven denaturing gradient gel electrophoresis (DGGE) bands were detected from gut of wood-feeding Nt, whereas 11 DGGE-bands were detected from that of phenol-feeding Nt. Out of 11 DGGE-bands, 5 of them were sequenced, and bacterial species including phenol-degrading bacteria were identified.     

Lack of mutagenic and toxic effects of low dose potassium bromate on kidneys in the Big Blue rat

Potassium bromate (KBrO3) has been classified as a genotoxic carcinogen based on positive results in the Ames test, and chromosome aberration and micronucleus tests. The purpose of the present study was to investigate the dose-response relationship for in vivo mutagenic and toxic effects of KBrO3 in the kidneys of Big Blue rats. In experiment 1, male Big Blue rats were divided into 8 groups. KBrO3 was dissolved in tap water and administered to groups 1-8 at concentrations of 0, 0.02, 0.2, 2, 8, 30, 125 and 500 ppm, respectively, for 16 weeks. Experiment 2 was performed to investigate the effects of KBrO3 at the 0.002 ppm dose approximately contained in the tap water on rat kidneys. Ten Big Blue rats were divided into 2 groups and given distilled water and tap water, respectively, for 16 weeks. In experiment 1, treatment with 500 ppm KBrO3 significantly increased the mutant and total mutation frequencies and frequency of GC to TA transversion of the lacI gene in the kidney compared to non-treatment control group, but 125 ppm and lower doses of KBrO3 had no effects. Histopathologically, renal toxic changes were observed in groups administered KBrO3 at 30 ppm or higher in a dose-dependent manner. PCNA positive cell indices in renal tubular cells were significantly increased in the kidney at doses of 125 and 500 ppm, but not at 30 ppm or lower doses, as compared to the control group. Furthermore, 8-hydroxy-2'-deoxyguanosine formation, a marker of oxidative stress, was significantly increased at 500 ppm. In experiment 2, there were no differences in any parameter between the distilled water and tap water groups. These results suggest the existence of no-effect levels for in vivo mutagenic and toxic effects, proliferation stimulus, and oxidative stress of KBrO3 in rat kidneys.     

Chemical modification of gamma-carboxyglutamic acid residues in prothrombin elicits a conformation similar to that of abnormal (des-gamma-carboxy)prothrombin

Chemical modification of gamma-carboxyglutamic acid (Gla) residues in human prothrombin to gamma-methyleneglutamic acid (gamma-MGlu) residues elicited a conformation similar, if not identical, to that of des-gamma-carboxy prothrombin or PIVKA-II, i.e., prothrombin molecules induced by vitamin K antagonists or vitamin K deficiency states. The reaction seems to proceed sequentially by preferentially modifying a Gla at residue 32 that is located innermost among 10 Gla residues of human prothrombin. The initial modification resulted in nearly 50% losses of barium salt adsorption, the procoagulant activity and thrombin generation by the prothrombinase complex. The subsequent modification of two Gla residues at positions 6 and 16 gave rise to the immunoreactivity to an established monoclonal antibody that specifically recognizes the des-gamma-carboxy prothrombin. Further modification of Gla residues increased the reactivity to the antibody, indicating that the conformation recognized by the antibody was stabilized so as to more readily fit the recognition site of the antibody. The appearance of the immunoreactivity was obviously related to the modification of Gla residues in prothrombin, since all other similarly treated derivatives of prothrombin lacking the Gla-domain failed to react with the antibody. Such chemically modified prothrombins may serve as models for studying abnormal des-gamma-carboxy prothrombin produced in vitamin K deficiency states.