Molecular cloning,nucleotide sequence and expression in Escherichia coli of the β-cyclodextrin glycosyltransferase gene from Bacillus circulans strain no. 8

Summary The -cyclodextrin glycosyltransferase (-CGTase) gene was isolated from a -library prepared from Bacillus circulans strain no. 8. It was subcloned into plasmid pTZ and expressed by its endogenous regulatory sequences in Escherichia coli JM 103. The structural gene was sequenced and showed an open reading frame for a polypeptide of 718 amino acid residues. The recombinant -CGTase had the same enzymatic properties as the extracellular CGTase (684 amino acid residues, corresponding to a mol. wt. of 74416) produced by B. circulans strain no. 8. The amino acid sequence showed the highest homology (74.6% identical amino acids) with the CGTase of B. circulans strain F-2, which had been erroneously described as an amylase. The homology with the enzyme from the alkalophilic Bacillus sp. strain no. 1011 was 71.4%. The amino acid sequence derived will be used for elucidating the three-dimensional structure of the enzyme. Offprint requests to: H. Bender     

Metal pollution recorded in extinctDreissena polymorpha communities, Lake Breitling, Havel Lakes system, Germany: a laser ablation inductively coupled plasma mass spectrometry study

Shells of probable former living communities ofDreissena polymorpha were found within sediments of the shallow polytrophic to hypertrophic hard water Lake Breitling (Havel-Lake system, Germany). Corresponding sediments have been deposited between approximately 1940 and 1970 and reflect increasing eutrophication and heavy metal pollution of the Lake during this period (Schettler, 1992). Single shells from various sediment depths were analysed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) along a line on the outer part of the shell. The response of these freshwater mussels to increasing heavy metal pollution is clearly reflected in the distribution of Pb, Cu, Cd and Zn within their valves. In general, Cd, Cu, Pb and Zn contents are lower, and the distribution more even, in the outer parts of the deepest (oldest) shells compared to shells from higher in the cored sediments. Notably higher contents of Cu, Pb and Zn were recorded from the central (umbonal) part of the more recent shells, but this behaviour is not recorded for Cd. Metabolic changes brought on by worsening environmental conditions are proposed to explain this phenomena. Acidity produced during anaerobic metabolism can be neutralised by dissolution of the carbonate part of the shell. Copper, Zn and Pb, which show an affinity for the organic component of the shell, may thus accumulate by repeated dissolution and reprecipitation of the shell during the lifetime of an individual organism. Cadmium, which is bound mainly in the aragonite of the shells, is released during the dissolution of carbonate and is not concentrated in the umbonal area of the shell.     

Anaerobic metabolism of 2-hydroxybenzoic acid (salicylic acid) by a denitrifying bacterium

The anaerobic metabolism of 2-hydroxybenzoic acid (salicylic acid) was studied in a denitrifying bacterium. Cells grown with 2-hydroxybenzoate were simultaneously adapted to degrade benzoate. Extract of these cells formed benzoate or benzoyl-CoA when incubated under reducing conditions with salicylate, MgATP, and coenzyme A, suggesting a degradation of 2-hydroxybenzoate via benzoate or benzoyl-CoA. This suggestion was supported by enzyme activity measurements. In extracts of 2-hydroxybenzoate-grown cells, the following enzyme activities were detected: two CoA ligases, one specific for 2-hydroxybenzoate, the other for benzoate, and two different enzyme activities catalyzing the reductive transformation of 2-hydroxybenzoyl-CoA. These findings suggest a degradation of salicylic acid by two new enzymes, 2-hydroxybenzoate-CoA ligase (AMP-forming) and 2-hydroxybenzoyl-CoA reductase (dehydroxylating), catalyzing (1) 2-hydroxybenzoate + MgATP + CoASH → 2-hydroxybenzoyl-CoA + MgAMP + PP_i (2) 2-hydroxybenzoyl-CoA + 2[H] → benzoyl-CoA + H₂O Benzoyl-CoA was dearomatized by reduction of the ring. This represents another case in which benzoyl-CoA is a central intermediate in anaerobic aromatic metabolism. Received: 1 February 1996 / Accepted: 24 February 1996     

PET1402, a nuclear gene required for proteolytic processing of cytochrome oxidase subunit 2 in yeast

The nuclear mutation pet ts1402 prevents proteolytic processing of the precursor of cytochrome oxidase subunit 2 (cox2) in Saccharomyces cerevisiae. The structural gene PET1402 was isolated by genetic complementation of the temperature-sensitive mutation. DNA sequence analysis identified a 1206-bp open reading frame, which is located 215 by upstream of the PET122 gene. The DNA sequence of PET1402 predicts a hydrophobic, integral membrane protein with four transmembrane segments and a typical mitochondrial targeting sequence. Weak sequence similarity was found to two bacterial proteins of unknown function. Haploid cells containing a null allelle of PET1402 are respiratory deficient.     

Carotenoids in the eyespot apparatus of the flagellate green alga Spermatozopsis similis: Adaptation to the retinal-based photoreceptor

Isolated intact eyespot apparatuses, the photoreceptive organelles involved in blue-light-mediated photoresponses of flagellate green algae, were analyzed regarding their carotenoid composition. Carotenoids from the eyespot apparatuses of Spermatozopsis similis were identified by high-performance liquid chromatography, visible-light absorption spectra, mass spectroscopy and by ¹H-nuclear magnetic resonance spectroscopy (carotenes), and compared with those of whole-cell extracts. Both extracts contained ,-carotene, ,-carotene (formerly -carotene), lycopene, lutein, zeaxanthin, violaxanthin and all-E-and 9-Z-neoxanthin. The relative carotenoid compositions, however, differed significantly. A twofold relative increase in the total carotene level was evident in the fraction enriched in eyespot apparatuses. This was mainly due to an increase in the monocyclic ,-carotene and the aliphatic lycopene, whereas the relative content of ,-carotene remained unchanged. On the other hand a relative decrease in the total xanthophyll content, especially of lutein and the epoxidic carotenoid neoxanthin, was observed in the eyespot apparatuses compared with the whole-cell extracts. The decrease of the latter resulted almost solely from a reduction of the 9-Z-rather than the all-E-isomer. The bulk of the carotenes is thought to be localized in the highly organized eyespot lipid globules, which act as a combined quarter-wave interference reflector and absorption screen for the photoreceptor in green algae. The enrichment of ,-carotene and lycopene in the eyespot apparatuses, extending the range of visible light absorption to longer wavelengths, represents an adaptation of the screen to the retinal-based photoreceptor of flagellate green algae and is one of the prerequisites for maximal directional sensitivity of the eyespot apparatus.Abbreviations ¹H-NMR nuclear magnetic resonance - IUPAC International Union of Pure and Applied Chemistry - VIS visible absorption spectra This work was supported by the Deutsche Forschungsgemeinschaft (G.K. and M.M.). M.G. was supported by a fellowship from the Norwegian Research Council of Science and Humanities.     

Modification of sialic acids by 9-O-acetylation is detected in human leucocytes using the lectin property of influenza C virus

Influenza C virus spike glycoprotein HEF specifically recognizesglycoconjugates containing 9-O-acetyl-N-acetylneuraminic acid.The same protein also contains an esterase activity. Takingadvantage of these two properties, influenza C virus was usedas a very sensitive probe for the detection of traces of 9-O-acetyl-N-acetylneuraminicacid in human leucocytes. The binding of influenza C virus toleucocyte glycoproteins and gangliosides separated by sodiumdodecyl sulphate–polyacrylamide gel electrophoresis andthin-layer chromatography, respectively, was assayed using achromogenic esterase substrate. In this way, glycoproteins ofB-lymphocytes and T-lymphocytes were found to contain 9-O-acetylatedsialic acids. Of the various 9-O-acetylated gangliosides detected,one had the characteristics of 9-O-acetylated G^D3. The identificationof 9-O-acetylated sialic acids on distinct glycoproteins andglycolipids should be helpful in assigning a physiological roleto this sugar. O-acetylation gangliosides influenza C virus lymphocytes sialic acids     

Spatial scale impacts microbial community composition and distribution within and across stream ecosystems in North and Central America

A mechanistic understanding of factors that structure spatiotemporal community composition is a major challenge in microbial ecology. Our study of microbial communities in the headwaters of three freshwater stream networks showed significant community changes at the small spatial scale of benthic habitats when compared to changes at mid- and large-spatial scales associated with stream order and catchment. Catchment (which included temperate and tropical catchments) had the strongest influence on community composition followed by habitat type (epipsammon or epilithon) and stream orders. Alpha diversity of benthic microbiomes resulted from interactions between catchment, habitat, and canopy. Epilithon contained relatively more Cyanobacteria and algae while Acidobacteria and Actinobacteria proportions were higher in epipsammic habitats. Turnover from replacement created ~60%–95% of beta diversity differences among habitats, stream orders, and catchments. Turnover within a habitat type generally decreased downstream indicating longitudinal linkages in stream networks while between habitat turnover also shaped benthic microbial community assembly. Our study suggests that factors influencing microbial community composition shift in dominance across spatial scales, with habitat dominating locally and catchment dominating globally.     

HPLC separation of the enantiomers of nicotine and nicotine-like compounds

A sensitive and reproducible HPLC method utilizing a commercially available chiral α₁-acid glycoprotein (AGP) phase has been developed to separate and quantify the enantiomers of nicotine. The method is suitable for routine use as indicated by column life. The quantification of (R/S:0.05/99.95)-nicotine or (R/S:99/1)-nicotine was possible. In addition, the separation or at least partial separation of the enantiomers of nornicotine and nornicotine-derived compounds was achieved. © 1993 Wiley-Liss, Inc.