Further development of the cassette-based pYC plasmid system by incorporation of the dominant hph, nat and AUR1-C gene markers and the lacZ reporter system

Dominant selection markers encoding hygromycin B phosphotransferase (hph), nourseothricin N-acetyltransferase (nat) and a mutant inositol phosphoceramide synthase (AUR1-C) were all incorporated into the pYC yeast plasmid vector system, thus expanding this system with possible alternatives to the use of G418 resistance. We found the markers to be of use not only in standard laboratory strains of Saccharomyces cerevisiae but also in an industrial strain of S. carlsbergensis (syn. of S. pastorianus) brewing yeast as well as in Saccharomyces kluyveri. As the pYC system contains means of counter-selection for plasmid loss and loop-out of integrated plasmids, it now provides ample opportunities for genetic manipulation of industrial and non-conventional yeasts when the URA3 marker and FOA counter-selection is not an option. Furthermore, the lacZ system for analyzing gene expression was included in the system.     

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.     

Endospores of thermophilic bacteria as tracers of microbial dispersal by ocean currents

Microbial biogeography is influenced by the combined effects of passive dispersal and environmental selection, but the contribution of either factor can be difficult to discern. As thermophilic bacteria cannot grow in the cold seabed, their inactive spores are not subject to environmental selection. We therefore conducted a global experimental survey using thermophilic endospores that are passively deposited by sedimentation to the cold seafloor as tracers to study the effect of dispersal by ocean currents on the biogeography of marine microorganisms. Our analysis of 81 different marine sediments from around the world identified 146 species-level 16S rRNA phylotypes of endospore-forming, thermophilic Firmicutes. Phylotypes showed various patterns of spatial distribution in the world oceans and were dispersal-limited to different degrees. Co-occurrence of several phylotypes in locations separated by great distances (west of Svalbard, the Baltic Sea and the Gulf of California) demonstrated a widespread but not ubiquitous distribution. In contrast, Arctic regions with water masses that are relatively isolated from global ocean circulation (Baffin Bay and east of Svalbard) were characterized by low phylotype richness and different compositions of phylotypes. The observed distribution pattern of thermophilic endospores in marine sediments suggests that the impact of passive dispersal on marine microbial biogeography is controlled by the connectivity of local water masses to ocean circulation.     

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.     

Expression and Cellular Distribution of Ubiquitin in Response to Injury in the Developing Spinal Cord of Monodelphis domestica

Ubiquitin, an 8.5 kDa protein associated with the proteasome degradation pathway has been recently identified as differentially expressed in segment of cord caudal to site of injury in developing spinal cord. Here we describe ubiquitin expression and cellular distribution in spinal cord up to postnatal day P35 in control opossums (Monodelphis domestica) and in response to complete spinal transection (T10) at P7, when axonal growth through site of injury occurs, and P28 when this is no longer possible. Cords were collected 1 or 7 days after injury, with age-matched controls and segments rostral to lesion were studied. Following spinal injury ubiquitin levels (western blotting) appeared reduced compared to controls especially one day after injury at P28. In contrast, after injury mRNA expression (qRT-PCR) was slightly increased at P7 but decreased at P28. Changes in isoelectric point of separated ubiquitin indicated possible post-translational modifications. Cellular distribution demonstrated a developmental shift between earliest (P8) and latest (P35) ages examined, from a predominantly cytoplasmic immunoreactivity to a nuclear expression; staining level and shift to nuclear staining was more pronounced following injury, except 7 days after transection at P28. After injury at P7 immunostaining increased in neurons and additionally in oligodendrocytes at P28. Mass spectrometry showed two ubiquitin bands; the heavier was identified as a fusion product, likely to be an ubiquitin precursor. Apparent changes in ubiquitin expression and cellular distribution in development and response to spinal injury suggest an intricate regulatory system that modulates these responses which, when better understood, may lead to potential therapeutic targets.     

Effect of CO2 on labelling of nerve and liver cells by nucleic acid precursors

In vivo experiments in mice demonstrated that 5% CO₂ content in the air inhaled did not change the labelling in autoradiograms from animals injected with [³H]uridine, [³H]orotic acid, [³H]hypoxanthine, [³H]lysine or [³H]cytidine. At 20% CO₂ content there was a significant decrease in labelling of brain cells with [³H]uridine and [³H]cytidine, but not following [³H]lysine; there was no labelling of nerve cells with [³H]orotic acid or [³H]hypoxanthine, but a control group was not included. The labelling of choroid plexus and hepatocytes was independent of the CO₂ concentration. A comparison of in vivo and in vitro experiments at 20% CO₂ content showed a similar significant decrease in labelling of brain cells with [³H]uridine and [³H]cytidine. It is concluded that a metabolic change is the most appropriate explanation of the CO₂ effect.     

Two Types of Endosymbiotic Bacteria in the Enigmatic Marine Worm Xenoturbella bocki

Two types of endosymbiotic bacteria were identified in the gastrodermis of the marine invertebrate Xenoturbella bocki (Xenoturbellida, Bilateria). While previously described Chlamydia-like endosymbionts were rare, Gammaproteobacteria distantly related to other endosymbionts and pathogens were abundant. The endosymbionts should be considered when interpreting the poorly understood ecology and evolution of Xenoturbella.Xenoturbella bocki is a benthic marine worm first described in 1949 and only found at a few geographical locations at apparently low population densities. It has a very simple body plan (Fig. ​(Fig.1A)1A) with a blind gut (cul-de-sac) and lacks coelomic cavities, a brain, and reproductive and excretory organs (35). Xenoturbella was originally classified as a flatworm, but molecular analyses have placed it within its own animal phylum, with various affiliations (reviewed in reference 34). The most recent phylogenetic analyses using mitochondrial genome and phylogenomic data indicate a position either within Deuterostomia (5) or at the base of Bilateria (14). Thus, the evolutionary history of Xenoturbella remains elusive and it is unclear whether its simple body plan represents a plesiomorphic character or, alternatively, is the result of secondary loss (34). In addition, the ecology of Xenoturbella is not well understood, leaving, e.g., its food source unresolved (4, 18).Open in a separate windowFIG. 1.(A) Schematic drawing of the simple body plan of Xenoturbella (redrawn from reference 35). (B) Cells of the gammaproteobacterial endosymbiont (red) detected by probe XenoGam441-CY3 at high magnification (scale bar, 2 μm). (C) Double hybridization of gammaproteobacterial and chlamydial endosymbionts with probes XenoGam441-CY3 (red) and Cps-1353mod-CY5 (displayed in yellow for better contrast, indicated by the arrow); blue-green structures, autofluorescent tissue recorded in the fluorescein isothiocyanate channel (scale bar, 5 μm). (D) FISH detection of gammaproteobacterial endosymbionts with probe XenoGam441-CY3 (red) in a cross section of an X. bocki specimen. The image is composed of five consecutive image stacks, spanning from the gut (to the very left) to the epidermis (to the very right) (magnification, ×400; scale bar, 50 μm). Dense clusters of gammaproteobacterial endosymbionts (red) are confined to the gastrodermis; 4′,6-diamidino-2-phenylindole (DAPI)-stained cell nuclei and mitochondria of X. bocki are displayed in blue; the green and yellow structures are autofluorescent tissue recorded in the fluorescein isothiocyanate channel. (E and F) TEM images of bacterial cells in sections of germinal tissue from a spermatid cluster (E) and of gastric tissue (F) of X. bocki. Arrows point to examples of different putative bacterial morphotypes. Scale bars, 0.5 μm. n, spermatid nucleus; m, spermatid mitochondrion.Symbiotic bacteria are found in a remarkable number of diverse marine invertebrates (11), where they affect both the ecology and the evolution of their hosts. The first indications of intracellular endosymbionts in Xenoturbella were published by Israelsson (17) and confirmed by electron microscopy of a Xenoturbella spermatid cluster (Fig. ​(Fig.1B);1B); however, our first molecular analyses identified the putative endosymbionts as Gammaproteobacteria, whereas Israelsson (17) had reported a group of endosymbiotic Chlamydiae. The objectives of this study were therefore (i) to identify the putative endosymbionts by 16S rRNA gene analysis, (ii) to localize them inside the host by fluorescence in situ hybridization (FISH), and (iii) to specifically test for the prevalence of the gammaproteobacterial endosymbionts in Xenoturbella.