Purification and characterisation of two extremely halotolerant xylanases from a novel halophilic bacterium

The present work reports for the first time the purification and characterisation of two extremely halotolerant endo-xylanases from a novel halophilic bacterium, strain CL8. Purification of the two xylanases, Xyl 1 and 2, was achieved by anion exchange and hydrophobic interaction chromatography. The enzymes had relative molecular masses of 43 kDa and 62 kDa and pI of 5.0 and 3.4 respectively. Stimulation of activity by Ca²⁺, Mn²⁺, Mg²⁺, Ba²⁺, Li²⁺, NaN₃ and isopropanol was observed. The K_m and V_max values determined for Xyl 1 with 4-O-methyl-d-glucuronoxylan are 5 mg/ml and 125,000 nkat/mg respectively. The corresponding values for Xyl 2 were 1 mg/ml and 143,000 nkat/mg protein. Xylobiose and xylotriose were the major end products for both endoxylanases. The xylanases were stable at pH 4–11 showing pH optima around pH 6. Xyl 1 shows maximal activity at 60°C, Xyl 2 at 65°C (at 4 M NaCl). The xylanases showed high temperature stability with half-lives at 60°C of 97 min and 192 min respectively. Both xylanases showed optimal activity at 1 M NaCl, but substantial activity remained for both enzymes at 5 M NaCl.Communicated by W.D. Grant     

Phylogenetic and functional diversity of bacteria in biofilms from metal surfaces of an alkaline district heating system

District heating systems (DHS) are extreme aqueous environments characterized by high temperatures, high pH (9.5-10.0), and low nutrient availability. Culture-independent and culture-dependent techniques showed that DHS may nevertheless harbour geno- and phenotypically diverse bacterial biofilm communities. Approximately 50% of the cells in biofilms growing on mild steel coupons in rotortorque reactors connected to the return line (40 degrees C) of a Danish DHS were detectable by FISH analysis and thus were probably metabolically active. A bacterial 16S rRNA gene clone library generated from the biofilms was dominated by proteobacterial phylotypes (closely related to known aerobic species) and by phylotypes affiliated to the anaerobic class Clostridia. Anoxic enrichment cultures derived from biofilms primarily contained 16S rRNA gene and dsrAB (encoding major subunits of dissimilatory sulfite reductase) phylotypes affiliated to the latter class. Alkalitolerant and neutrophilic anaerobic bacteria were isolated from the DHS, including novel Gram-positive and deltaproteobacterial sulfate-reducers and sulfite-reducers constituting novel Gram-positive lineages. In total, 39 distinct 16S rRNA gene phylotypes representing ten classes were identified. The detection of several alkalitolerant, sulfide-producing, and, thus, potentially biocorrosive species underlines the need to maintain a high water quality in the DHS in order to prevent the proliferation of these species.     

Salinity and temperature effects on accessibility of soluble and cross-linked insoluble xylans to endo-xylanases

Different responses to salinity were observed for an extremely halotolerant endo-xylanase when assayed with soluble birchwood glucoronoxylan and cross-linked dyed insoluble birchwood glucoronoxylan. Shrinking of insoluble xylan particles due to increased ionic strength is proposed as the explanation. Temperature affected the xylanase activity measurement on the insoluble xylan greatly, likely due to increased enzyme accessible surface of the substrate at high temperatures.     

Elucidation of the origin of multiple conformations of the human α3-chain type VI collagen C-terminal Kunitz domain: The reorientation of the Trp21 ring

Summary The human 3-chain type VI collagen C-terminal Kunitz domain fragment (3(VI)) has been studied by two-dimensional ¹H–¹H and ¹H–¹³C NMR spectroscopy at 303 K. It is shown that the secondary structure of the protein is strikingly similar to that of BPTI, and that a number of unusual H chemical shifts, which are highly conserved in Kunitz-domain proteins, are also observed for 3(VI). Further-more a series of exchange cross peaks observed in ¹H–¹H spectra shows that a large number of protons in the central -sheet exist in two different chemical environments, corresponding to two unequally populated conformations that are slowly exchanging on the NMR time scale. Several protons, including Ser⁴⁷⁽⁵³⁾ H, Arg³²⁽³⁸⁾ H², and Gln⁴⁸⁽⁵⁴⁾ H², all located in the vicinity of the Trp²¹⁽²⁷⁾ ring in the crystal structure of 3(VI) [Arnoux, B. et al. (1995) J. Mol. Biol., 246, 609–617], have very different chemical shifts in the two conformations, the most affected being Gln⁴⁸⁽⁵⁴⁾ H² (=1.53 ppm), which is placed directly above the Trp²¹⁽²⁷⁾ ring in the crystal structure of 3(VI). It is concluded that the origin of the multiple conformations of the central -sheet is a reorientation of the Trp²¹⁽²⁷⁾ ring. From the intensities of corresponding signals in the two conformations, the population of the minor conformation was found to be 6.4±0.2% of that of the major conformation, while a rate constant k_M=1.01±0.05 s^-1 for the major to minor interconversion was obtained from a series of NOESY spectra with different mixing times. In addition, it is shown that Cys¹⁴⁽²⁰⁾-Cys³⁸⁽⁴⁴⁾ disulfide bond isomerization, previously observed in BPTI [Otting, G. et al. (1993) Biochemistry, 32, 3570–3582], is also likely to occur in 3(VI).     

Endothelin secretion is regulated by cyclic AMP and phosphatase 2A in endothelial cells

Endothelin is a 21 amino acid peptide secreted by endothelial cells and is the most potent vasoconstrictor known. The present study examines regulatory mechanisms of endothelin secretion, focusing on the role of protein phosphorylation. Endothelin secretion was measured by radioimmunoassay in primary cultures of human umbilical vein endothelial cells. While treatment that raised cAMP levels reduced the basal endothelin secretion rate, agents that elevated cGMP had no effect. Downregulation or inhibition of protein kinase C resulted in decreased endothelin secretion, suggesting that protein kinase C regulates endothelin secretion in the opposite direction to cAMP dependent protein kinases Okadaic acid, at concentrations that selectively inhibit protein phosphatases 2A, reduced the endothelin secretion and the effects of okadaic acid and db-cAMP were additive. Endothelin production was stimulated by fetal calf serum and by the protein kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7), but was inhibited by the calmodulin antagonist trifluoperazine. The present findings that regulators of cAMP-dependent protein kinases, protein kinase C, calmodulin, and protein phosphatase 2A all affect endothelin secretion suggest that endothelin secretion is controlled by phosphorylation/dephosphorylation of as yet unidentified regulatory proteins within the cell. © 1994 Wiley-Liss, Inc.     

Initial establishment of vegetation in a man-made coastal area in Denmark

A man-made coastal area (8 km long, 300 m wide), constructed from seed-free, marine sand was investigated from 1978 to 1983 as regards relief, soil properties, and establishment of vegetation.
The following developments were apparent:

• 1. 

  The outer shore zone changed from a Cakile -dominated vegetation to a sparse and less diverse vegetation.
• 2. 

  The inner shore zone maintained spontaneous Ammophila as dominant and developed into a mobile dune with changing species.
• 3. 

  The outer dune slope with planted Ammophila developed into a mobile dune dominated by vigorous Ammophila.
• 4. 

  The dune zone with planted Ammophila: The outer part developed towards a somewhat fixed dune with increasing diversity. The inner part developed into a diverse and more fixed dune community with mainly Festuca rubra.
• 5. 

  The inner dune slope developed into a vegetation comprising few species.
• 1. 

  The grassland zone, initially sown with Festuca rubra and Lolium perenne , changed into a grass-herb-vegetation dominated by Festuca rubra , with a slowly increasing number of immigrating species, changing from annuals to perennials, in particular species of Fabaceae and Hippophaë.

     

Endothelial inositol phosphate generation and prostacyclin production in response to G-protein activation by AlF4-.

In order to elucidate the role of guanine-nucleotide-binding proteins (G-proteins) in endothelial prostacyclin (PGI2) production, human umbilical vein endothelial cells, prelabelled with either [3H]inositol or [3H]arachidonic acid, were stimulated with the non-specific G-protein activator aluminium fluoride (AlF4-). AlF4- caused a dose- and time-dependent generation of inositol phosphates, release of arachidonic acid and production of PGI2. The curves for the three events were similar. When the cells were stimulated in low extracellular calcium (60 nM), they released [3H]arachidonic acid and produced PGI2, but depleting the intracellular Ca2+ stores by pretreatment with the Ca2+ ionophore A23187 totally inhibited both events, although the cells still responded when extracellular Ca2+ was added. The Ca2+ ionophore did not inhibit the generation of inositol phosphates in cells maintained at low extracellular Ca2+. Pertussis toxin pretreatment (14 h) altered neither inositol phosphate nor PGI2 production in response to AlF4-. To investigate the functional role of the diacylglycerol/protein kinase C arm of the phosphoinositide system, the cells were pretreated with the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) or the protein kinase C inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7). TPA inhibited the AlF4(-)-induced inositol phosphate generation but stimulated both the release of arachidonic acid and the production of PGI2. H7 had opposite effects both on inositol phosphate generation and on PGI2 production. These results suggest that AlF4(-)-induced PGI2 production is mediated by a pertussis-toxin-insensitive G-protein which activates the phosphoinositide second messenger system. This production of PGI2 can be modulated by protein kinase C activation, both at the level of inositol phosphate generation and at the level of arachidonic acid release.     

Recolonization of experimentally bared soil in a grazed common in Denmark

In an old grazed and very diverse common in Central Zealand, Denmark, the recolonization of vegetation on experimentally bared mineral soil was studied over a six years period (198691). In six experimental squares (1×1 m) in pairs placed in three different areas the plant cover and 10 cm of top soil was removed in 1986 after an analysis (Hult-Sernander-Du Rietz method) of the vegetation in a central, fixed plot (50 × 50 cm) and an examination of the flora in the nearest surroundings (<10 m).
In each of the following years (1987–91) the recolonizating vegetation of the bared plots was analysed again. After one year an almost closed vegetation was already established in most of the plots. The new vegetation consists mostly of immigrating previously found species but often with another cover value. A small number of the original species are still absent after five years. A smaller number of the species in the new vegetation are intrusive, and most of these species are coming from the nearest surroundings. In the first five years all recolonization is by means of diaspores, and the diversity increases in the last years. The paper discusses returning, disappearing and new intrusive species with background in their way of dispersal of diaspores. The conclusion is that in 1991 a succession is still - but slowly - going on and that a totally stable vegetation possibly never will be established.