Phylogenetic analysis and characterization of lipolytic activity of halophilic archaeal isolates.

Five isolates designated as B45, D83A, A206A, A85 and E49 and found to possess a activity were taxonomically classified on the basis of their phylogenetic, phenotypic and chemotaxonomic characteristics. The isolates were determined to be Gram-negative, catalase and oxidase positive, hydrolyzing Tween 80 and 60 but not starch, need 3.5-4 M NaCl for optimal growth and lack of anaerobic growth with arginine or DMSO. All isolates had the highest lipolytic activity at pH 8.5. Lipase and esterase activities increased with salt concentration up to 3-4.5 M NaCl, and decreased at 5 M NaCl. Esterase and lipase showed their maximal activities at 50-55 degrees C and 60-65 degrees C, respectively. The phylogenetic tree constructed by the neighbor-joining method indicated that the strain B45 and A85 were closely related to the members of genera Halovivax and Natrinema, respectively. The closest relative of the strain A206A and D83A were found to be Haloterrigena saccharevitans. The strain E49 displayed a more distant relationship to known strains.

Isolation of alpha-glucosidase-producing thermophilic bacilli from hot springs of Turkey.

From 42 different hot springs in 6 provinces belonging to distinct geographical regions of Turkey, 451 thermophilic bacilli were isolated and 67 isolates with a high amylase activity were selected to determine the alpha-glucosidase production capacities by using pNPG as a substrate. Alpha-glucosidase production capacities of the isolates varied within the range from 77.18 to 0.001 U/g. Eleven of our thermophilic bacilli produced alpha-glucosidase at significant levels comparable with that of the reference strains tested, thus five strains, F84b (77.18 U/g), A333 (48.64 U/g), F84a (36.64 U/g), E134 (32.09 U/g), and A343 (10.79 U/g) were selected for further experiments. 16S rDNA sequence analysis revealed that these selected isolates all belonged to thermophilic bacilli 16S rDNA genetic group 5, four of them representing the genus Geobacillus, while strain A343 had an uncultured bacterium as the closest relative. Changes of alpha-glucosidase levels in the intracellular and extracellular fractions were determined during 48-h cultivation of A333, A343, F84a, F84b, E134, and the reference strain G. stearothermophilus ATCC 12980. According to alpha-glucosidase production type and enzyme levels in intracellular and extracellular fractions, Geobacillus spp. A333, F84a and F84b were defined as extracellular enzyme producers, whereas the thermophilic bacterium A343 was found to be an intracellular alpha-glucosidase producer, similar to ATCC 12980 strain. Geobacillus sp. E134 differed in alpha-glucosidase production type from all tested isolates and the reference strain; it was described as a membrane-associated cell-bound enzyme producer. In this study, apart from screening a great number of new thermophilic bacilli from the hot springs of Turkey, which have not yet been thoroughly studied, five new thermostable alpha-1,4-glucosidase-producing bacilli that have biotechnological potential with alpha-glucosidases located at different cell positions were obtained.

[Characterization of extremely halophilic archaea isolated from saline environment in different parts of Turkey].

Ninety-five extremely halophilic strains were isolated from six distinct saline regions of Turkey by using complex medium containing 25% NaCl. The selected regions are Tuz Golu (salt lake), Ankara; Aci Lake, Denizli; Salda Lake, Denizli; Seyfe Lake, Kirsehir; Tuzla Lake, Kayseri; and Bolluk Lake, Konya. The isolated strains were tested for motility, Gram reaction, cell and colony morphologies, pigmentation, biochemical characteristics, and antibiotic sensitivities. According to membrane glycerol diether moieties and antibiotic susceptibilities, all isolated strains were found to belong to the domain Archaea. All isolates were examined for the presence of plasmids by agarose gel electrophoresis and it was established that most isolates contained plasmids that varied in number and whose molecular sizes ranged from 1 to 36.9 kbp. Whole-cell protein profiles from isolates were analyzed by SDS-PAGE and a similarity dendogram was constructed using the UPGMA method. Significant similarities and differences were observed among the isolates. The strains were clustered in eight groups and ten of our isolates were placed in the same group with the standard strains. The current study represents the first isolation and characterization of such a large collection of archeal strains from Turkey.

Bacteriocin diversity in Bacillus sphaericus.

Nondenaturing polyacrylamide gel electrophoresis revealed the presence of diversity among bacteriocins produced by strains of Bacillus sphaericus. Bacteriocin bands of six strains (pathogenic and non pathogenic) were found to be located just below the stacking gel. However, in two other strains (1 pathogenic and 1 collection strain) more than one protein band with bacteriocin activity were seen in the middle of resolving gel. In bacteriocin-treated cultures, electron-microscopy studies revealed the growth of lysedswollen ghost cells, and loss of viability among sensitive strains.

[Comparison of Staphylococcus spp. cellular and extracellular proteins by SDS-PAGE]. / Sravnenie Staphylococcus spp. po belkovym profiliam posle denaturiruiushchego élektroforeza kletochnykh i vnekletochnykh belkov v polyakrilamidnom gele.

In this study, a total of fifteen staphylococcal strains belonging to different species were characterized by whole-cell and extracellular protein profiles using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results are presented as dendrograms after quantitative analysis of the band patterns with a computer program. Visual inspection of protein bands and cluster analysis of protein patterns of 15 strains representing 10 Staphylococcus species showed that whole-cell and extracellular protein profiles differed in several protein bands in Staphylococcus aureus, S. epidermidis, S. simulans and other species of Staphylococcus; however, the differences were insufficient for reliable differentiation of Staphylococcus species by the SDS-PAGE method.

Effects of UV-light on Bacillus sphaericus and its protection by chemicals.

Although when UV-irradiated seven most toxic strains of Bacillus sphaericus lost their viabilities between 2.5-4.5 min, their larvicidal activity was protected for longer periods. Benzaldehyde, cinnamaldehyde, salicylaldehyde, methylene blue and yeast extract showed good protective effect for spore viability and larvicidal activity from UV inactivation in B. sphaericus. This protective effect has also been confirmed by SDS-PAGE analyses whereby the 42 kDa and 51 kDa toxic proteins bands did not disappear following UV treatment.

Electrophysiological effects of Bacillus sphaericus binary toxin on cultured mosquito cells.

The electrophysiological effects of both trypsinactivated native and 42- and 51-kDa cloned binary toxins of Bacillus sphaericus were investigated on cultured Culex quinquefasciatus cells using the patchclamp technique. Rates of reduction in whole-cell membrane resistance were correlated with increasing native toxin concentration. The 42- or 51-kDa cloned toxin alone at 50 micrograms/ml reduced the resistance. Electrophysiological effects occurred before any changes were visible by phase-contrast microscopy.

Dissemination of a single clone of methicillin-resistant Staphylococcus aureus among Turkish hospitals.

A collection of 39 methicillin-resistant Staphylococcus aureus (MRSA) stains derived from six different hospitals in Ankara and one hospital in Barsa, Turkey, were analyzed by multiple genotyping. In agreement with the other genotyping assays, pulsed-field gel electrophoresis of DNA macrorestriction fragments identified genetic homogeneity among all MRSA isolates studies. It is concluded that a major clone of MRSA has spread through a large part of Turkey, causing longitudinally persistent colonization in all of the institutions surveyed.

Maize genotypes classified as null at the glu locus have beta-glucosidase activity and immunoreactive protein.

Maize beta-glucosidase (beta-D-glucoside glucohydrolase; EC 3.2.1.21) was extracted from coleoptiles of 15 maize genotypes (3 normals, 10 nulls, and 2 hybrids) in two fractions, the soluble and the insoluble. The enzyme activity was measured spectrophotometrically in the soluble fraction and also studied on zymograms after native gel electrophoresis and isoelectric focusing. The enzyme was purified from a normal genotype by anion-exchange chromatography and preparative electrophoresis. Antisera were raised in four rabbits, and the soluble and the insoluble extracts of each genotype were analyzed for a cross-reacting material by ELISA and immunoblotting. The results showed that extracts from both the normal and the null genotypes had beta-glucosidase activity, and the activity measured spectrophotometrically was 2- to 10-fold higher in normals than in nulls. Zymograms of the null genotypes were devoid of distinct bands that were present in those of normals and hybrids from crosses between normals and nulls. Zymograms of both the normal and the null genotypes had a diffuse, smeared zone of activity at the cathodic end of native gels. A cross-reacting antigen was present in extracts of both genotypes when assayed by ELISA and a 60-kD polypeptide (beta-glucosidase monomer) was detected by four different monospecific beta-glucosidase antisera on Western blots by immunostaining. Moreover, six of seven null genotypes had a larger amount of their 60-kD polypeptide in the insoluble fraction than in the soluble fraction. These data show that both the null and the normal genotypes have similar amounts of the enzyme protein, but the enzyme occurs mostly as insoluble or poorly soluble polymers in nulls, and the monogenic inheritance reported for the null alleles of the glu locus is likely to be for a factor encoded by another locus which affects directly or indirectly the solubility of the enzyme by increasing its polymerization into large quaternary structures.