Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78

White rot fungi efficiently degrade lignin, a complex aromatic polymer in wood that is among the most abundant natural materials on earth. These fungi use extracellular oxidative enzymes that are also able to transform related aromatic compounds found in explosive contaminants, pesticides and toxic waste. We have sequenced the 30-million base-pair genome of Phanerochaete chrysosporium strain RP78 using a whole genome shotgun approach. The P. chrysosporium genome reveals an impressive array of genes encoding secreted oxidases, peroxidases and hydrolytic enzymes that cooperate in wood decay. Analysis of the genome data will enhance our understanding of lignocellulose degradation, a pivotal process in the global carbon cycle, and provide a framework for further development of bioprocesses for biomass utilization, organopollutant degradation and fiber bleaching. This genome provides a high quality draft sequence of a basidiomycete, a major fungal phylum that includes important plant and animal pathogens.     

Molecular basis of Colorado potato beetle adaptation to potato plant defence at the level of digestive cysteine proteinases

Potato synthesises high levels of proteinase inhibitors in response to insect attack. This can adversely affect protein digestion in the insects, leading to reduced growth, delayed development and lowered fecundity. Colorado potato beetle overcomes this defence mechanism by changing the composition of its digestive proteinases. The induced cysteine proteinases in the adapted gut sustain a normal rate of protein hydrolysis either by inactivating the inhibitors by cleavage or by insensitivity to the inhibitors as a result of high Kis. In this study cDNA clones of cysteine proteinases in adapted guts were isolated by nested PCR on the basis of N-terminal sequences previously determined for purified enzymes (Gruden et al., 2003). The cysteine proteinase cDNAs can be classified into three groups: intestains A, B and C. The amino acid identity is more than 91% within and 35-62% between the groups. They share 43-50% identity to mammalian cathepsins S, L, K, H, J and cathepsin-like enzymes from different arthropods. Homology modelling predicts that intestains A, B and C follow the general fold of papain-like proteinases. Intestains from each group, however, differ in some specific structural characteristics in the S1 and S2 binding sites that could influence enzyme-inhibitor interaction and thus, provide different mechanisms of resistance to inhibitors for the different enzymes. Gene expression analysis revealed that the intestains A and C, but not B, are induced twofold by potato plants with high levels of proteinase inhibitors.     

Terpenoid metabolism in wild-type and transgenic Arabidopsis plants

Volatile components, such as terpenoids, are emitted from aerial parts of plants and play a major role in the interaction between plants and their environment. Analysis of the composition and emission pattern of volatiles in the model plant Arabidopsis showed that a range of volatile components are released, primarily from flowers. Most of the volatiles detected were monoterpenes and sesquiterpenes, which in contrast to other volatiles showed a diurnal emission pattern. The active terpenoid metabolism in wild-type Arabidopsis provoked us to conduct an additional set of experiments in which transgenic Arabidopsis overexpressing two different terpene synthases were generated. Leaves of transgenic plants constitutively expressing a dual linalool/nerolidol synthase in the plastids (FaNES1) produced linalool and its glycosylated and hydroxylated derivatives. The sum of glycosylated components was in some of the transgenic lines up to 40- to 60-fold higher than the sum of the corresponding free alcohols. Surprisingly, we also detected the production and emission of nerolidol, albeit at a low level, suggesting that a small pool of its precursor farnesyl diphosphate is present in the plastids. Transgenic lines with strong transgene expression showed growth retardation, possibly as a result of the depletion of isoprenoid precursors in the plastids. In dual-choice assays with Myzus persicae, the FaNES1-expressing lines significantly repelled the aphids. Overexpression of a typical cytosolic sesquiterpene synthase resulted in the production of only trace amounts of the expected sesquiterpene, suggesting tight control of the cytosolic pool of farnesyl diphosphate, the precursor for sesquiterpenoid biosynthesis. This study further demonstrates the value of Arabidopsis for studies of the biosynthesis and ecological role of terpenoids and provides new insights into their metabolism in wild-type and transgenic plants.     

The Slc11a1 (Nramp1) gene controls efficacy of mycobacterial treatment of allergic asthma

Genes controlling antibacterial resistance may be important in the hygiene hypothesis, which states that lack of bacterial infections during childhood would favor development of allergic disease. We, therefore, studied whether Nramp1 (Slc11a1) alleles, which determine susceptibility (Nramp1(s)) or resistance (Nramp1(r)) to intracellular bacteria, affect the efficacy of heat-killed Mycobacterium vaccae in the treatment of allergic asthma in a mouse model. Treatment of OVA-sensitized Nramp1(s) mice with M. vaccae suppressed airway hyperresponsiveness, airway eosinophilia, Ag-specific IgE, and IL-4 and IL-5 production after OVA aerosol challenge. In contrast, M. vaccae hardly affected these parameters in Nramp1(r) mice. In addition, The Nramp1 gene affected both T cell-mediated responses to M. vaccae in vivo and the level of macrophage activation after stimulation with M. vaccae in vitro. In conclusion, the efficacy of M. vaccae in preventing allergic and asthmatic manifestations in a mouse model is strongly affected by Nramp1 alleles. These findings could have important implications for the future use of mycobacteria and their components in the prevention or treatment of allergic asthma. A new link is described between genes, the environment, and the development of allergy, in which the Nramp1 gene fine tunes the hygiene hypothesis.     

Immobilization of the distal hinge in the labile serpin plasminogen activator inhibitor 1: identification of a transition state with distinct conformational and functional properties

The serpin plasminogen activator inhibitor-1 (PAI-1) plays an important role in the regulation of the fibrinolytic activity in blood. In plasma, PAI-1 circulates mainly in the active conformation. However, PAI-1 spontaneously converts to a latent conformation. This conversion comprises drastic conformational changes in both the distal and the proximal hinge region of the reactive center loop. To study the functional and conformational rearrangements associated solely with the mobility of the proximal hinge, disulfide bonds were introduced to immobilize the distal hinge region. These mutants exhibited specific activities comparable with that of PAI-1-wt. However, the engineered disulfide bond had a major effect on the conformational and associated functional transitions. Strikingly, in contrast to PAI-1-wt, inactivation of these mutants yielded a virtually complete conversion to a substrate-like conformation. Comparison of the digestion pattern (with trypsin and elastase) of the mutants and PAI-1-wt revealed that the inactivated mutants have a conformation differing from that of latent and active PAI-1-wt. Unique trypsin-susceptible cleavage sites arose upon inactivation of these mutants. The localization of these exposed residues provides evidence that a displacement of alphahF has occurred, indicating that the proximal hinge is partly inserted between s3A and s5A. In conclusion, immobilization of the distal hinge region in PAI-1 allowed the identification of an "intermediate" conformation characterized by a partial insertion of the proximal hinge region. We hypothesize that locking PAI-1 in this transition state between active and latent conformations is associated with a displacement of alphahF, subsequently resulting in substrate behavior.     

Complementary impact of paralogous Oxa1-like proteins of Bacillus subtilis on post-translocational stages in protein secretion

In mitochondria, chloroplasts, and Gram-negative eubacteria, Oxa1p(-like) proteins are critical for the biogenesis of membrane proteins. Here we show that the Gram-positive eubacterium Bacillus subtilis contains two functional Oxa1p orthologues, denoted SpoIIIJ and YqjG. The presence of either SpoIIIJ or YqjG is required for cell viability. Whereas SpoIIIJ is required for sporulation, YqjG is dispensable for this developmental process. The stability of two membrane proteins was found to be mildly affected upon SpoIIIJ limitation in the absence of YqjG. Surprisingly, the topology and stability of other membrane proteins remained unaffected under these conditions. In contrast, SpoIIIJ- and YqjG-limiting conditions resulted in a strong post-translocational defect in the stability of secretory proteins. Together, these data indicate that SpoIIIJ and YqjG of B. subtilis are involved in both membrane protein biogenesis and protein secretion. However, the reduced stability of secretory proteins seems to be the most prominent phenotype of SpoIIIJ/YqjG-depleted B. subtilis cells. In conclusion, our observations show that SpoIIIJ and YqjG have different, but overlapping functions in B. subtilis. Most importantly, it seems that different members of the Oxa1p protein family have acquired at least partly distinct, species-specific, functions that are essential for life.     

Twitch and tetanic tension during culture of mature Xenopus laevis single muscle fibres

Investigation of the mechanisms of muscle adaptation requires independent control of the regulating factors. The aim of the present study was to develop a serum-free medium to culture mature single muscle fibres of Xenopus laevis. As an example, we used the culture system to study adaptation of twitch and tetanic force characteristics, number of sarcomeres in series and fibre cross-section. Fibres dissected from m. iliofibularis (n = 10) were kept in culture at a fibre mean sarcomere length of 2.3 microm in a culture medium without serum. Twitch and tetanic tension were determined daily. Before and after culture the number of sarcomeres was determined by laser diffraction and fibre cross-sectional area (CSA) was determined by microscopy. For five fibres twitch tension increased during culture and tetanic tension was stable for periods varying from 8 to 14 days ('stable fibres'), after which fibres were removed from culture for analysis. Fibre CSA and the number of sarcomeres in series remained constant during culture. Five other fibres showed a substantial reduction in twitch and tetanic tension within the first five days of culture ('unstable fibres'). After 7-9 days of culture, three of these fibres died. For two of the unstable fibres, after the substantial force reduction, twitch and tetanic tension increased again. Finally at day 14 and 18 of culture, respectively, the tensions attained values higher than their original values. For stable fibres, twitch contraction time, twitch half-relaxation time and tetanus 10%-relaxation time increased during culture. For unstable fibres these parameters fluctuated. For all fibres the stimulus threshold fluctuated during the first two days, and then remained constant, even for the fibres that were cultured for at least two weeks. It is concluded that the present culture system for mature muscle fibres allows long-term studies within a well-defined medium. Unfortunately, initial tetanic and twitch force are poor predictors of the long-term behaviour of the fibres.     

Discovering lactic acid bacteria by genomics

This review summarizes a collection of lactic acid bacteria that are now undergoing genomic sequencing and analysis. Summaries are presented on twenty different species, with each overview discussing the organisms fundamental and practical significance, nvironmental habitat, and its role in fermentation, bioprocessing, or probiotics. For those projects where genome sequence data were available by March 2002, summaries include a listing of key statistics and interesting genomic features. These efforts will revolutionize our molecular view of Gram–positive bacteria, as up to 15 genomes from the low GC content lactic acid bacteria are expected to be available in the public domain by the end of 2003. Our collective view of the lactic acid bacteria will be fundamentally changed as we rediscover the relationships and capabilities of these organisms through genomics.     

Cationic interactions at the human dopamine transporter reveal binding conformations for dopamine distinguishable from those for the cocaine analog 2 alpha-carbomethoxy-3 alpha-(4-fluorophenyl)tropane

In membrane preparations, CFT, a phenyltropane cocaine analog, and dopamine (DA) interact with the recombinant human dopamine transporter (hDAT) in Na+ -free medium. Na+ markedly increased the transporter's affinity for CFT, but had little or no effect on DA potency for inhibiting CFT binding. Raising [Na+ ] from 20 to 155 mm reduced Li+ -induced increase in DA K (i), but not CFT K (d). The presence of 155 mm Na+ enhanced the tolerance to low pH of CFT Kd but not DA Ki. Leucine substitution for tryptophan 84 (W84L) in transmembrane domain (TM) 1 or asparagine substitution for aspartate 313 (D313N) in TM 6 did not or only modestly enhance the affinity of Na+ -independent CFT binding, and retained the near normal ability of DA, Li+, K+, or H+ to inhibit this binding. However, the mutations significantly enhanced the Na+ stimulation of CFT binding as well as the Na+ antagonism against Li+ and H+ inhibition of CFT binding. In contrast, the mutations neither changed the Na+ -insensitive feature of DA Ki nor enhanced the Na+ protection of DA Ki against Li+ 's inhibitory effect, though they caused Na+ protection of DA Ki against H+ 's inhibitory action. These results are consistent with the existence of binding conformations for DA that are distinguishable from those for CFT, and with a differential association of cation interactions with DA and CFT binding. The mutations likely alter Na+ -bound state(s) of hDAT, preferentially strengthening the positive allosteric coupling between Na+ and CFT binding, and reducing the impact of Li+ or H+ on the CFT binding.