Transposition of Tn1831 to sym plasmids of Rhizobium leguminosarum and Rhizobium trifolii

Abstract All transposon-induced symbiotic mutants of Rhizobium described so far have been obtained using Tn 5 , which codes for kanamycin resistance (Km^R). To enable genetic complementation studies, we tried to find an effective transposon carrying another resistance marker. We report here a method for the apparent random transposition in Rhizobium of Tn 1831 , which codes for resistance against spectinomycin (Sp), streptomycin (Sm) and mercury chloride. When the suicide plasmid pMP12 (RP4::Tn 1831 , Km::Mu) was transferred to Rhizobium , in almost all cases the exconjugants harbour a deleted transfer-deficient R plasmid. From this deleted R plasmid transposition occurred to self-transmissible Sym-plasmids of R. leguminosarum and R. trifolii . Using this method a number of Tn 1831 -induced symbiotic mutants of pRL1JI were isolated.     

Biocontrol traits of Pseudomonas spp. are regulated by phase variation

Of 214 Pseudomonas strains isolated from maize rhizosphere, 46 turned out to be antagonistic, of which 43 displayed clear colony phase variation. The latter strains formed both opaque and translucent colonies, designated as phase I and phase II, respectively. It appeared that important biocontrol traits, such as motility and the production of antifungal metabolites, proteases, lipases, chitinases, and biosurfactants, are correlated with phase I morphology and are absent in bacteria with phase II morphology. From a Tn5luxAB transposon library of Pseudomonas sp. strain PCL1171 phase I cells, two mutants exhibiting stable expression of phase II had insertions in gacS. A third mutant, which showed an increased colony phase variation frequency was mutated in mutS. Inoculation of wheat seeds with PCL1171 bacteria of phase I morphology resulted in efficient suppression of take-all disease, whereas disease suppression was absent with phase II bacteria. Neither the gacS nor the mutS mutant was able to suppress take-all, but biocontrol activity was restored after genetic complementation of these mutants. Furthermore, in a number of cases, complementation by gacS of wild-type phase II sectors to phase I phenotype could be shown. A PCL1171 phase I mutant defective in antagonistic activity appeared to have a mutation in a gene encoding a lipopeptide synthetase homologue and had lost its biocontrol activity, suggesting that biocontrol by strain PCL1171 is dependent on the production of a lipopeptide. Our results show that colony phase variation plays a regulatory role in biocontrol by Pseudomonas bacteria by influencing the expression of major biocontrol traits and that the gacS and mutS genes play a role in the colony phase variation process. Therefore phase variation not only plays a role in escaping animal defense but it also appears to play a much broader and vital role in the ecology of bacteria producing exoenzymes, antibiotics, and other secondary metabolites.     

1α,25-Dihydroxyvitamin D3-induced down-regulation of the checkpoint proteins, Chk1 and Claspin, is mediated by the pocket proteins p107 and p130

A previous cDNA microarray analysis in murine MC3T3-E1 osteoblasts revealed a cluster of genes involved in cell cycle progression that was significantly down-regulated after a single treatment with 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] [L. Verlinden, G. Eelen, I. Beullens, M. Van Camp, P. Van Hummelen, K. Engelen, R. Van Hellemont, K. Marchal, B. De Moor, F. Foijer, H. Te Riele, M. Beullens, M. Bollen, C. Mathieu, R. Bouillon, A. Verstuyf, Characterization of the condensin component Cnap1 and protein kinase Melk as novel E2F target genes down-regulated by 1,25-dihydroxyvitamin D3, J. Biol. Chem. 280 (45) (2005) 37319–37330]. Among those genes were the DNA replication and DNA damage checkpoint proteins, Chk1 and Claspin, of which the human homologues were recently shown to be E2F-responsive. Quantitative real-time PCR experiments in 1,25(OH)₂D₃-treated MC3T3-E1 cells confirmed the down-regulation observed in the microarray experiment. Moreover, Chk1 and Claspin promoter activities were also reduced after incubation with 1,25(OH)₂D₃, and this reduction was mediated through the E2F recognition motifs within their promoters because mutation of these motifs almost completely abolished the repressive effect of 1,25(OH)₂D₃. The antiproliferative effect of 1,25(OH)₂D₃ as well as its potential to down-regulate the expression of Chk1 and Claspin depended on the pocket proteins p107 and p130 because 1,25(OH)₂D₃ lost its antiproliferative action and failed to repress these E2F-target genes in p107^−/−;p130^−/−-cells, but not in pRb^−/−-cells.     

Parkinson’s Protein α-Synuclein Binds Efficiently and with a Novel Conformation to Two Natural Membrane Mimics

Binding of human α-Synuclein, a protein associated with Parkinson’s disease, to natural membranes is thought to be crucial in relation to its pathological and physiological function. Here the binding of αS to small unilamellar vesicles mimicking the inner mitochondrial and the neuronal plasma membrane is studied in situ by continuous wave and pulsed electron paramagnetic resonance. Local binding information of αS spin labeled by MTSL at positions 56 and 69 respectively shows that also helix 2 (residues 50–100) binds firmly to both membranes. By double electron-electron resonance (DEER) on the mutant spin labeled at positions 27 and 56 (αS 27/56) a new conformation on the membrane is found with a distance of 3.6 nm/ 3.7 nm between residues 27 and 56. In view of the low negative charge density of these membranes, the strong interaction is surprising, emphasizing that function and pathology of αS could involve synaptic vesicles and mitochondria.     

The Rcs Two-Component System Regulates Expression of Lysozyme Inhibitors and Is Induced by Exposure to Lysozyme

The Escherichia coli Rcs regulon is triggered by antibiotic-mediated peptidoglycan stress and encodes two lysozyme inhibitors, Ivy and MliC. We report activation of this pathway by lysozyme and increased lysozyme sensitivity when Rcs induction is genetically blocked. This lysozyme sensitivity could be alleviated by complementation with Ivy and MliC.In gram-negative bacteria, the cell envelope represents an important functional compartment that extends from the cytoplasmic membrane to the outer membrane and supports a number of essential processes, such as solute transport, protein translocation, and respiratory energy generation (15). In addition, the cell envelope accommodates the bacterial peptidoglycan layer, a distinct and structurally vital element of the cell. Most recently, Laubacher and Ades (10) have demonstrated that the Rcs phosphorelay system of Escherichia coli, originally described as regulator of capsule synthesis, is activated by β-lactam antibiotics that inhibit penicillin-binding proteins and consequently interfere with peptidoglycan synthesis. Moreover, mutational activation of the Rcs pathway provided significant protection against these antibiotics, indicating that members of this regulon can prevent or repair the peptidoglycan damage caused by β-lactam antibiotics (10).Interestingly, ivy and ydhA, two genes encoding specific lysozyme inhibitors, were found to reside under this Rcs regulon (8, 10). Ivy (inhibitor of vertebrate lysozyme, formerly known as YkfE) was discovered in 2001 as the first bacterial lysozyme inhibitor (1, 14), while the inhibitory activity of YdhA was only recently revealed by our research group (3). Although Ivy and YdhA are both able to inhibit c-type lysozymes, such as human lysozyme and hen egg white lysozyme (HEWL), they are structurally unrelated (1, 16). Interestingly, YdhA belongs to a group of proteins with a common conserved COG3895 domain that are widely spread among the Proteobacteria (3, 16). Unlike Ivy, which resides in the periplasm, YdhA is a lipoprotein and was therefore renamed MliC (membrane-bound lysozyme inhibitor of c-type lysozyme) (3).Given the elementary observation that the two currently known lysozyme inhibitors of E. coli are both part of the Rcs regulon that can in turn be induced by antibiotic-mediated peptidoglycan stress, we wondered whether Rcs induction could also result from exposure to lysozyme itself. To test this, we introduced a tolA knockout from MG1655 tolA (3) into strain DH300 that is equipped with a genomic rprA-lacZ fusion able to report Rcs activation (12), in order to increase outer membrane permeability for HEWL (Table ​(Table11 lists all strains). A stationary-phase culture of the resulting strain, designated LC100, was diluted 1/100 in 4 ml fresh LB medium with different final concentrations of HEWL (0, 5, 10, 25, and 50 μg/ml), and after 2.5 h of further growth at 37°C, β-galactosidase activity was measured (13). Interestingly, rprA-lacZ was significantly induced at HEWL concentrations of >10 μg/ml, up to 4.4-fold at 50 μg/ml (Fig. ​(Fig.1A).1A). This induction could be completely abolished upon the additional introduction of a knockout of rcsB (strain LC102), the response regulator required to activate gene expression in the Rcs pathway. Moreover, knocking out rcsF (strain LC101), the outer membrane lipoprotein sensor that triggers the Rcs pathway upon antibiotic-mediated peptidoglycan stress (10), also resulted in a loss of lysozyme induction. As a comparison, rprA-lacZ induction in DH300 treated with amdinocillin (Sigma-Aldrich, Bornem, Belgium), as previously described (10), resulted in a 16-fold increase in β-galactosidase activity (Fig. ​(Fig.1B).1B). Please note that the difference in basal β-galactosidase levels between LC100 and DH300 (Fig. 1A and B) is probably due to the tolA mutation in LC100, which is known to result in a higher basal expression of the Rcs pathway (5). These data clearly demonstrate that the Rcs phosphorelay can indeed be activated by exposure to lysozyme and that this induction is mediated by the outer membrane sensor rcsF. This also implies that the Rcs pathway responds to different types of peptidoglycan stress, as β-lactam antibiotics block the formation of peptide side-chain cross-links by binding irreversibly to the transpeptidases, while lysozyme hydrolyzes the heteropolysaccharide backbone.Open in a separate windowFIG. 1.Induction of the Rcs pathway in LC100 (tolA::Kn Rcs⁺) with different HEWL concentrations (0 to 50 μg/ml) (A) and in DH300 (Rcs⁺) with (+) or without (−) amdinocillin treatment (B). Rcs induction is measured as β-galactosidase activity originating from a genomic rprA-lacZ reporter fusion and expressed in Miller units (13). Error bars indicate standard deviations of results from three replicate experiments. The corresponding RcsB⁻ strain (LC102) and the RcsF⁻ strain (LC101) showed rprA-lacZ inductions of <10 Miller units when subjected to lysozyme treatments and are therefore not shown.

TABLE 1.

Bacterial strains and plasmids used in the study

Identification of a functional Antigen5-related allergen in the saliva of a blood feeding insect,the tsetse fly

Our previous screening of a Glossina morsitans morsitans λgt11 salivary gland expression library with serum of a tsetse fly exposed rabbit identified a cDNA encoding Tsetse Antigen5 (TAg5, 28.9 kDa), a homologue of Antigen5 sting venom allergens. Recombinant TAg5 was produced in Sf9 cells in order to assess its immunogenic properties in humans. Plasma from a patient that previously exhibited anaphylactic reactions against tsetse fly bites contained circulating anti-TAg5 and anti-saliva IgEs. In a significant proportion of plasma samples of African individuals, TAg5 and saliva binding IgEs (respectively 56 and 65%) can be detected. Saliva, harvested from flies that were subjected to TAg5-specific RNA interference (RNAi), displayed significantly reduced IgE binding potential. Allergenic properties of TAg5 and tsetse fly saliva were further illustrated in immunized mice, using an immediate cutaneous hypersensitivity and passive cutaneous anaphylaxis assay. Collectively, TAg5 was illustrated to be a tsetse fly salivary allergen, demonstrating that Antigen5-related proteins are represented as functional allergens not only in stinging but also in blood feeding insects.     

The Conservation Value of Traditional Rural Landscapes: The Case of Woodpeckers in Transylvania,Romania

Land use change is a major threat to global biodiversity. Forest species face the dual threats of deforestation and intensification of forest management. In regions where forests are under threat, rural landscapes that retain structural components of mature forests potentially provide valuable additional habitat for some forest species. Here, we illustrate the habitat value of traditional wood pastures for a woodpecker assemblage of six species in southern Transylvania, Romania. Wood pastures are created by long-term stable silvo-pastoral management practices, and are composed of open grassland with scattered large, old trees. Because of their demanding habitat requirements, woodpeckers share habitat with many other bird species, and have been considered as possible indicator species for bird species diversity. We first compared woodpecker assemblages between forests and wood pastures. Second, we grouped features of wood pastures into three spatial contexts and addressed how these features related to the occurrence of three woodpecker species that are formally protected. Woodpecker species composition, but not the number of species, differed between forests and wood pastures, with the green woodpecker occurring more commonly in wood pastures, and the lesser spotted woodpecker more commonly in forests. Within wood pastures, the intermediate context (especially surrounding forest cover) best explained the presence of the grey-headed and middle spotted woodpecker. By contrast, variables describing local vegetation structure and characteristics of the surrounding landscape did not affect woodpecker occurrence in wood pastures. In contrast to many other parts of Europe, in which several species of woodpeckers have declined, the traditional rural landscape of Transylvania continues to provide habitat for several woodpecker species, both in forests and wood pastures. Given the apparent habitat value of wood pastures for woodpeckers we recommend wood pastures be explicitly considered in relevant policies of the European Union, namely the Habitats Directive and the EU Common Agricultural Policy.     

The folliculostatins of two dipteran insect species, their relation to matrix proteins and prospects for practical applications

The first insect folliculostatic peptide was isolated from vitellogenic ovaries of the mosquitoAedes aegypti. This decapeptide directly inhibits trypsin biosynthesis in the gut, and indirectly ovarian development. The factor was named Trypsin Modulating Oostatic Factor or TMOF by its discoverers. From the fleshfly Neobellieria bullata 2 folliculostatins have been isolated, the hexapeptide Neb-TMOF and the 19-mer Neb-colloostatin. The available data suggest that at least 2 of the 3 folliculostatins originate from matrix (like) proteins present in the ovary, a hitherto unknown source of hormones. Furthermore, one of the folliculostatins (Neb-TMOF) is a potent inhibitor of ecdysone biosynthesis by larval ring glands of fleshflies. The discovery of the dipteran folliculostatins, which do not show any resemblance to inhibins of vertebrates, may significantly contribute to a better understanding of the hormonal control of growth in insects and perhaps, in other animals as well. None of the 3 folliculostatins is blocked at its N- or C-terminus. This, in combination with the pleiotropy of their effects and the narrow species specificity make such peptides prime candidates for, testing their potential in insect pest control by means of molecular biological methods.