Production of a thermophilic maltooligosyl-trehalose synthase in <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

The thermoacidophilic archaeon Sulfolobus solfataricus MT4 encodes a maltooligosyltrehalose synthase (MTS), that catalyzes an intramolecular transglycosylation process converting the glycosidic linkages at the reducing end of dextrins from alpha-1,4 into alpha-1,1. In this research the gene encoding MTS was cloned and expressed in Lactococcus lactis NZ9000 using the so-called NICE system. Growth conditions of the recombinant strain were optimized in flask experiments in relation to enzyme production. Batch experiments in 2 L-fermenters were performed on the best identified semidefined medium and 256 U L(-1) of recombinant MTS were produced. Purified recombinant MTS shows its optimal activity at 70 degrees C and pH 5.5, prefers maltoheptaose and maltohexaose as substrates, and demonstrates minimal side hydrolytic activity.     

ATP hydrolysis and pristinamycin IIA inhibition of the Staphylococcus aureus Vga(A), a dual ABC protein involved in streptogramin A resistance

In Gram-positive bacteria, a large subfamily of dual ATP-binding cassette proteins confers acquired or intrinsic resistance to macrolide, lincosamide, and streptogramin antibiotics by a far from well understood mechanism. Here, we report the first biochemical characterization of one such protein, Vga(A), which is involved in streptogramin A (SgA) resistance among staphylococci. Vga(A) is composed of two nucleotide-binding domains (NBDs), separated by a charged linker, with a C-terminal extension and without identified transmembrane domains. Highly purified Vga(A) displays a strong ATPase activity (K(m) = 78 mum, V(m) = 6.8 min(-1)) that was hardly inhibited by orthovanadate. Using mutants of the conserved catalytic glutamate residues, the two NBDs of Vga(A) were shown to contribute unequally to the total ATPase activity, the mutation at NBD2 being more detrimental than the other. ATPase activity of both catalytic sites was essential for Vga(A) biological function because each single Glu mutant was unable to confer SgA resistance in the staphylococcal host. Of great interest, Vga(A) ATPase was specifically inhibited in a non-competitive manner by the SgA substrate, pristinamycin IIA (PIIA). A deletion of the last 18 amino acids of Vga(A) slightly affected the ATPase activity without modifying the PIIA inhibition values. In contrast, this deletion reduced 4-fold the levels of SgA resistance. Altogether, our results suggest a role for the C terminus in regulation of the SgA antibiotic resistance mechanism conferred by Vga(A) and demonstrate that this dual ATP-binding cassette protein interacts directly and specifically with PIIA, its cognate substrate.     

Genetic relatedness of <Emphasis Type="Italic">Trichoderma</Emphasis> isolates antagonistic against <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">dianthi</Emphasis> inflicting carnation wilt

Twenty-eight isolates of Trichoderma belonging to four different species were screened in vitro for their antagonistic ability against Fusarium oxysporum f.sp. dianthi causing carnation wilt. Three different levels of antagonism observed in dual plate assay were further confirmed by cell-free culture filtrate experiments. Isolates showing class I level of antagonism produced maximum lytic enzymes, chitinases and beta-1,3-glucanases. Genetic variability of 25 selected isolates was assessed by random amplified polymorphic DNA technique and the amplified products were correlated for their level of antagonism. Unweighed pair-group method with arithmetical averages cluster analysis revealed prominent inter-and intraspecific genetic variation among the isolates. Based on their genetic relationship, the isolates were mainly distributed into 3 major groups representing T. atroviride, T. pseudokoningii and T. harzianum, with 20-35% interspecific dissimilarity. However, the polymorphism shown by the isolates did not correlate to their level of antagonism.     

Seasonal distribution and dive behaviour of harp seals (<Emphasis Type="Italic">Pagophilus groenlandicus</Emphasis>) of the White Sea–Barents Sea stock

Eight adult female harp seals (Pagophilus groenlandicus) of the White Sea–Barents Sea stock were tagged with satellite-linked dive recorders during the nursing period and followed from breeding in late February 1995 until moulting in late April 1995. Another ten adult harp seals of both sexes were tagged and followed from moult in early May 1996 until breeding in late February the following year. Between breeding and moult the seals were distributed along the coasts of Kola of Russia and eastern Finnmark of Norway, coinciding in time and space with the spawning capelin (Mallotus villosus). Between moulting and breeding they encircled the entire Barents Sea, mostly in open water, using the water column from 20 to 300 m, and in so doing by and large reflecting the annual migrations of the capelin. Capelin is therefore assumed to be the main source of prey for the White Sea–Barents Sea stock of harp seals, to be substituted, in part, by amphipods (e.g. Themisto libellula) in mid-summer and polar cod (Boreogadus saida) and herring (Clupea pallasii) in late autumn and winter. These data provide a baseline for the evaluation of the effects of future climatic change in the rich Barents Sea ecosystem.     

Characterisation of mutations in 77 patients with X-linked myotubular myopathy,including a family with a very mild phenotype

X-linked myotubular myopathy is characterised by neonatal hypotonia, muscle weakness and respiratory distress in affected males, leading often to early death, although prolonged survival is observed in milder forms, or as a result of prolongation of ventilation support. It is caused by mutations in the MTM1 gene, which encodes a phosphatase called myotubularin, which has been highly conserved during evolution, down to yeasts ( S. cerevisiae and S. pombe). To date, 251 mutations have been identified in unrelated families, corresponding to 158 different disease-associated mutations, which are widespread throughout the gene. We have found additional mutations in 77 patients, including 35 novel ones. We identified a missense mutation N180K in a 67-year-old grandfather (the oldest known patient with an MTM1 mutation), previously suspected to have autosomal centronuclear myopathy, and in his two grandsons also mildly affected. Mild and moderate phenotypes associated with novel missense mutations and with a translation initiation defect mutation are discussed, as well as severe phenotypes associated with particular novel mutations. With the present report, 192 different mutations in the MTM1 gene have been described in 328 families. The spectrum of mutations is now enlarged from the very severe classic neonatal phenotype to very mild phenotype allowing survival to the age of 67 years.     

The effect of crop rotation of six forest tree species on peat-bark substrate enzymatic activity

The paper focuses on the effect of a nine-year utilisation of the peat-bark substrate and crop rotation of six main forest tree species on changes in the substrate enzymatic activity during successive rotation cycles. The study was conducted in the forest nursery in the years 1989–1997. Seedlings of Scots pine Pinus sylvestris, Norway spruce Picea abies, European larch Larix decidua, pendiculate oak Quercus robur, common beech Fagus silvatica, and silver birch Betula overrucosa were grown on peat-bark substrate. The activity of soil enzymes: betaglucosidase, invertase, urease, asparginase, acid phosphatase and dehydrogenases was assessed. The succession of three 3-year crop rotation cycles with species following each other according to the rotation plan was subject to observations. The obtained results have confirmed recent suppositions that the tree species and their rotation modify soil enzymatic activity. The enzymatic activity of the peat-bark substrate changed after each three-year crop rotation cycle and decreased with time. After the second crop rotation cycle the activity of betaglucosidase, urease, asparginase was found to be lower, and the activity of invertase and dehydrogenases — higher. After three crop rotation cycles the positive effect of appropriate species rotation on the enzymatic activity of the substrate was noted.     

Vasa expression and germ-cell specification in the spider mite <Emphasis Type="Italic">Tetranychus urticae</Emphasis>

The specification of germ cells is an important process during the development of all animals. Expression of an evolutionarily conserved gene such as vasa can be used as a marker for germ cell fate. We have isolated a vasa-related gene from the two-spotted spider mite (Tetranychus urticae) and used it to examine the segregation of germ cells in this animal. In spider mites, vasa expression first appears in a group of cells that do not join the initial blastoderm surface. Instead, these cells remain in the interior of the blastoderm and then migrate to posterior regions of the embryo, where they form a cluster that appears in regions of the embryo consistent with the gonads. The expression pattern of this spider mite vasa homologue implies a novel process acts to specify germ cells in this species and that the specification of germ cells is an evolutionarily labile process.     

Expression and localization of human lysozyme in the endosperm of transgenic rice

In order to understand the characteristics of recombinant protein expression and sublocalization in rice ( Oryza sativa L.) endosperm, we examined the expression level of human lysozyme protein and its subcellular location in transgenic rice seeds driven by rice glutelin and globulin promoters and signal peptides. A time course of human lysozyme expression during endosperm development was analyzed. The results showed that the expression profile of recombinant protein accumulation in endosperm paralleled that of the two storage proteins. Immunofluorescence microscopy revealed that human lysozyme and storage proteins co-localized to type-II protein bodies. Both promoter-signal peptide parings targeted recombinant protein to the protein bodies. In addition, a transgenic line with a higher lysozyme expression level exhibited morphologically different protein bodies with an unbalanced composition of lysozyme and native storage proteins. The high-level expression of recombinant protein distorted the trafficking and sorting of native storage proteins in rice endosperm and affected the expression of native storage protein.     

Potential effects of physiological plastic responses to salinity on population networks of the estuarine crab <Emphasis Type="Italic">Chasmagnathus granulata</Emphasis>

Chasmagnathus granulata is a South American crab occurring in estuarine salt marshes of the Brazilian, Uruguayan and Argentine coasts. Life history is characterized by an export strategy of its larval stages. I reviewed information on experimental manipulation of salinity during embryonic and larval development (pre- and posthatching salinities), and on habitat characteristics of C. granulata in order to determine potential effects of larval response to salinity in the field and to suggest consequences for the population structure. Local populations are spread over coastal areas with different physical characteristics. Benthic phases occupy estuaries characterized by different patterns of salinity variation, and release larvae to coastal waters characterized by strong salinity gradients. The zoea 1 of C. granulata showed a strong acclimatory response to low salinity. This response operated only during the first weeks of development (during zoeae 1 and 2) since subsequent larval survival at low posthatching salinities was consistently low. Larvae developing at low salinity frequently followed a developmental pathway with five instead of four zoeal stages. The ability to acclimate and the variability in larval development (i.e. the existence of alternative developmental pathways) could be interpreted as a strategy to buffer environmental variability at spatial scales of local or population networks. Early survivorship and production of larvae may be relatively high across a rather wide range of variability in salinity (5–32‰). Plastic responses to low salinity would therefore contribute to maintain a certain degree of population connectivity and persistence regardless of habitat heterogeneity. Electronic Publication