Synthesis of the membrane fusion and hemagglutinin proteins of measles virus, using a novel baculovirus vector containing the beta-galactosidase gene.

An improved baculovirus expression vector was developed to expedite screening and facilitate oligonucleotide-directed mutagenesis. This vector contained twin promoters derived from the P10 and polyhedrin genes of Autographica californica nuclear polyhedrosis virus. The P10 promoter directed the synthesis of beta-galactosidase, whereas the polyhedrin promoter controlled the synthesis of foreign gene products. These two genes recombined with wild-type virus genome to yield recombinants which were polyhedrin negative, produced the foreign gene product, and formed blue plaques when beta-galactosidase indicator was present in the agarose overlay. An origin of replication derived from M13 or f1 bacteriophage was also included in the plasmid to permit the synthesis of single-stranded DNA. This template DNA was used to introduce or delete sequences through the process of site-specific mutagenesis. The measles virus virion possesses a membrane envelope which contains two glycoproteins: the hemagglutinin (H) and membrane fusion (F) proteins. The H polypeptide has receptor-binding and hemagglutinating activity, whereas the F protein mediates virus penetration of the host cell, formation of syncytia, and hemolysis of erythrocytes. Genes for these two glycoproteins were inserted into the NheI cloning site of the modified expression vector described above. The vector and purified wild-type viral DNA were introduced into Sf9 insect cells by calcium phosphate precipitation. A mixture of wild-type and recombinant virus was generated and used to infect Sf9 cells, which were subsequently overlaid with agarose. After 3 days, 0.1 to 1% of the plaques became blue in the presence of beta-galactosidase indicator. At least 70% of these blue viral colonies contained the foreign gene of interest as determined by dot blot analysis. Recombinant virus was separated from contaminating wild-type virus through several rounds of plaque purification. Insect cells were then infected with the purified recombinants, and synthesis of H and F proteins were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot detection and Coomassie blue staining. Glycosylation of the proteins appeared to be impaired somewhat, and the precursor to the F protein was not completely cleaved by the proteases present in insect host cells. On the other hand, both proteins appeared to be active in hemagglutination, hemolysis, and cell fusion assays. Levels of synthesis were in the order of 50 to 150 mg of protein per 10(8) cells.     

Identification,Purification, and Characterization of Transpeptidase and Glycosyltransferase Domains of Streptococcus pneumoniae Penicillin-Binding Protein 1a

Resistance to β-lactam antibiotics in Streptococcus pneumoniae is due to alteration of penicillin-binding proteins (PBPs). S. pneumoniae PBP 1a belongs to the class A high-molecular-mass PBPs, which harbor transpeptidase (TP) and glycosyltransferase (GT) activities. The GT active site represents a new potential target for the generation of novel nonpenicillin antibiotics. The 683-amino-acid extracellular region of PBP 1a (PBP 1a*) was expressed in Escherichia coli as a GST fusion protein. The GST-PBP 1a* soluble protein was purified, and its domain organization was revealed by limited proteolysis. A protease-resistant fragment spanning Ser 264 to Arg 653 exhibited a reactivity profile against both β-lactams and substrate analogues similar to that of the parent protein. This protein fragment represents the TP domain. The GT domain (Ser 37 to Lys 263) was expressed as a recombinant GST fusion protein. Protection by moenomycin of the GT domain against trypsin degradation was interpreted as an interaction between the GT domain and the moenomycin.The synthesis of the bacterial cell wall requires cytoplasmic and periplasmic enzymes. The final steps of peptidoglycan biosynthesis occur outside the cytoplasmic membrane, and they are catalyzed by membrane-bound penicillin-binding proteins (PBPs). PBPs play essential roles in cell division and morphology (6, 20, 31). Based upon their molecular sizes and amino acid sequence similarities, PBPs can be classified into two groups (6): low-molecular-weight (low-M_r) PBPs, which act as d,d-carboxypeptidases, and high-molecular-weight (high-M_r) PBPs, which carry transpeptidase (TP) and glycosyltransferase (GT) activities. The high-M_r group can be further divided into bifunctional enzymes with TP and GT activities (class A) and monofunctional TP enzymes (class B).β-Lactam antibiotics bind with high affinity specifically to d,d-carboxypeptidase and TP domains because of their structural similarity to the natural substrates, the stem peptides. This binding results in the formation of a covalent acyl-PBP enzyme complex, leading to the inactivation of PBPs.High-M_r PBPs are multidomain proteins (6). The three-dimensional structure of Streptococcus pneumoniae PBP 2x (class B high-M_r PBP) illustrates this domain organization (25). The only non-penicillin-binding domain of known function is the GT domain, corresponding to the N-terminal region of class A PBPs. This GT activity, clearly identified in Escherichia coli PBP 1b, is difficult to measure (23, 29, 31–35). It is insensitive to penicillin but sensitive to moenomycin, an antibiotic which is not used for human therapy (23, 29, 32, 33).S. pneumoniae is one of the major human pathogens of the upper respiratory tract, causing pneumonia, meningitis, and ear infections. Six PBPs have been identified in S. pneumoniae: high-M_r PBPs 1a, 1b, 2a, 2x, and 2b and low-M_r PBP 3 (8). PBPs 1a, 1b, and 2a belong to class A, while PBPs 2x and 2b are monofunctional class B proteins. Deletion of pbp2x and pbp2b in S. pneumoniae is lethal for the bacteria, while the deletion of pbp1a is tolerated (11), probably due to compensation by PBP 1b. This has been observed for E. coli class A PBP 1a, whose deletion can be compensated for by PBP 1b (36). In clinical isolates of resistant pneumococci, pbp1a, pbp2x, and pbp2b genes were shown to present a mosaic organization, encoding PBPs with reduced affinity for β-lactam antibiotics (2, 5, 15, 18). The specific resistance to ceftriaxone and cefotaxime of S. pneumoniae from the hospital environment is mediated by modification of PBP 2x and PBP 1a (22). Furthermore, gene transfer of pbp1a, pbp2x, and pbp2b from resistant strains conferred penicillin resistance on sensitive S. pneumoniae strains under laboratory conditions (2–4, 14, 15, 27, 30).The effort to overcome resistance to antibiotics in S. pneumoniae might therefore benefit from a detailed understanding of the molecular basis of TP and GT activities. The GT domain represents a new potential target for novel nonpenicillin antibiotics. Here, we delineate the GT and TP domains of S. pneumoniae PBP 1a* (a water-soluble form of PBP 1a) by limited proteolytic digestion and expression of recombinant domains. The TP activity of PBP 1a* and that of the isolated TP domain were compared. We also present evidence for an interaction between the isolated GT domain and moenomycin.     

EFFECTS OF SMALL-SCALE TURBULENCE ON PHOTOSYNTHESIS,PIGMENTATION, CELL DIVISION,AND CELL SIZE IN THE MARINE DINOFLAGELLATE GOMAULAX POLYEDRA (DINOPHYCEAE)1

Several experiments were conducted to understand better the physiological mechanisms underlying growth inhibition of the dinoflagellate Gonyaulax polyedra Stein due to small-scale turbulence shear. To measure photosynthetic ¹⁴C uptake, a “phytoplankton wheel” device for rotating cultures in closed bottles was used. Turbulence was quantified biologically in the bottles by comparing growth inhibition with that in cultures with constant shear between a fixed cylinder and an outer concentric rotating cylinder (a stable Couette flow). At saturating irradiances, particulate photosynthesis (P_sat) or photosynthesis per unit chlorophyll (P^B_sat) were not inhibited completely at the highest turbulence level (26.6 rad.s^?1), and photosynthesis was less sensitive than growth. Photosynthesis per cell (P^C_sat) was increased by turbulence. In three experiments on the effects of turbulence on photosynthesis versus irradiance curves, the slope of the curve, α, for particulate photosynthesis at limiting irradiances did not change. Photosynthesis per unit chlorophyll per unit irradiance (α^B) decreased at high (but not intermediate) turbulence levels. Photosynthesis per cell per unit irradiance, α^C, increased with turbulence, suggesting an increase in photosynthetic efficiency in turbulent cultures. In two of the three experiments, respiration rates increased with turbulence, and in one experiment excretion of photosynthetically fixed ¹⁴C was not affected by motion. Ratios of accessory pigments to chlorophyll a did not change with turbulence, but pigments per cell and per dry weight increased with turbulence. These findings suggest little or no disruption of the photosynthetic apparatus. When turbulence was applied for 1 week, β-carotene increased while peridinin and diadinoxanthin decreased, suggesting inhibition of synthesis of these latter pigments by prolonged turbulence. Since cell numbers did not increase or decreased during turbulent 72–h incubations, cell division was inhibited and also the cells were very much enlarged. Increases in α^C per cell suggest that, in the sea, photo synthetic metabolism can persist efficiently without cell division during turbulent episodes. After turbulence ceases or reaches low levels again, cells can then divide and blooms may form. Thus, blooms can come or go fairly rapidly in the ocean depending on the degree of wave- and wind-induced turbulence.     

The human metapneumovirus matrix protein stimulates the inflammatory immune response in vitro

Each year, during winter months, human Metapneumovirus (hMPV) is associated with epidemics of bronchiolitis resulting in the hospitalization of many infants. Bronchiolitis is an acute illness of the lower respiratory tract with a consequent inflammation of the bronchioles. The rapid onset of inflammation suggests the innate immune response may have a role to play in the pathogenesis of this hMPV infection. Since, the matrix protein is one of the most abundant proteins in the Paramyxoviridae family virion, we hypothesized that the inflammatory modulation observed in hMPV infected patients may be partly associated with the matrix protein (M-hMPV) response. By western blot analysis, we detected a soluble form of M-hMPV released from hMPV infected cell as well as from M-hMPV transfected HEK 293T cells suggesting that M-hMPV may be directly in contact with antigen presenting cells (APCs) during the course of infection. Moreover, flow cytometry and confocal microscopy allowed determining that M-hMPV was taken up by dendritic cells (moDCs) and macrophages inducing their activation. Furthermore, these moDCs enter into a maturation process inducing the secretion of a broad range of inflammatory cytokines when exposed to M-hMPV. Additionally, M-hMPV activated DCs were shown to stimulate IL-2 and IFN-γ production by allogeneic T lymphocytes. This M-hMPV-mediated activation and antigen presentation of APCs may in part explain the marked inflammatory immune response observed in pathology induced by hMPV in patients.     

The full-length Streptococcus pneumoniae major pilin RrgB crystallizes in a fibre-like structure, which presents the D1 isopeptide bond and provides details on the mechanism of pilus polymerization

RrgB is the major pilin which forms the pneumococcal pilus backbone. We report the high-resolution crystal structure of the full-length form of RrgB containing the IPQTG sorting motif. The RrgB fold is organized into four distinct domains, D1-D4, each of which is stabilized by an isopeptide bond. Crystal packing revealed a head-to-tail organization involving the interaction of the IPQTG motif into the D1 domain of two successive RrgB monomers. This fibrillar assembly, which fits into the electron microscopy density map of the native pilus, probably induces the formation of the D1 isopeptide bond as observed for the first time in the present study, since neither in published structures nor in soluble RrgB produced in Escherichia coli or in Streptococcus pneumoniae is the D1 bond present. Experiments performed in live bacteria confirmed that the intermolecular bond linking the RrgB subunits takes place between the IPQTG motif of one RrgB subunit and the Lys183 pilin motif residue of an adjacent RrgB subunit. In addition, we present data indicating that the D1 isopeptide bond is involved in RrgB stabilization. In conclusion, the crystal RrgB fibre is a compelling model for deciphering the molecular details required to generate the pneumococcal pilus.     

Production of low‐Cs+ rice plants by inactivation of the K+ transporter OsHAK1 with the CRISPR‐Cas system

The occurrence of radiocesium in food has raised sharp health concerns after nuclear accidents. Despite being present at low concentrations in contaminated soils (below μm ), cesium (Cs⁺) can be taken up by crops and transported to their edible parts. This plant capacity to take up Cs⁺ from low concentrations has notably affected the production of rice (Oryza sativa L.) in Japan after the nuclear accident at Fukushima in 2011. Several strategies have been put into practice to reduce Cs⁺ content in this crop species such as contaminated soil removal or adaptation of agricultural practices, including dedicated fertilizer management, with limited impact or pernicious side‐effects. Conversely, the development of biotechnological approaches aimed at reducing Cs⁺ accumulation in rice remain challenging. Here, we show that inactivation of the Cs⁺‐permeable K⁺ transporter OsHAK1 with the CRISPR‐Cas system dramatically reduced Cs⁺ uptake by rice plants. Cs⁺ uptake in rice roots and in transformed yeast cells that expressed OsHAK1 displayed very similar kinetics parameters. In rice, Cs⁺ uptake is dependent on two functional properties of OsHAK1: (i) a poor capacity of this system to discriminate between Cs⁺ and K⁺; and (ii) a high capacity to transport Cs⁺ from very low external concentrations that is likely to involve an active transport mechanism. In an experiment with a Fukushima soil highly contaminated with ¹³⁷Cs⁺, plants lacking OsHAK1 function displayed strikingly reduced levels of ¹³⁷Cs⁺ in roots and shoots. These results open stimulating perspectives to smartly produce safe food in regions contaminated by nuclear accidents.     

Ultrastructure des photorécepteurs de Lineus ruber (O. F. Müller) (Hétéronémertes Lineïdae)

Summary The eyes of the Nemertean worm Lineus ruber are situated on the dorsal face of the animal in two lateral bands in front of the cerebral ganglia. There are 6 or 8 ocelli per animal enclosed in the cephalic parenchyme. They consist of a pigmented eyecup in which 50 photoreceptor cells are present. The photoreceptrice cells have:dendritic processes which are full of mitochondria, neurotubules, vacuoles and only one axial filament;a pericaryon with rough endoplasmic reticulum, a Golgi apparatus and few mitochondria.

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Résumé Les yeux de la Némerte marine Lineus ruber sont situés sur la face dorsale de l'animal, en deux rangées parallèles à l'avant des ganglions cérébroïdes. On en dénombre 6 à 8 localisés dans le parenchyme céphalique. Ils consistent en une cupule pigmentaire entourant une cinquantaine de cellules visuelles qui rejoignent les centres nerveux supérieurs. Pour les cellules photoréceptrices on distingue:un processus dendritique riche en mitochondries, neurotubules, vacuoles et possédant un axe médian;un corps cellulaire où l'on trouve du réticulum endoplasmique rugueux, un appareil de Golgi développé mais peu de mitochondries.

     

Functional characterization of penicillin-binding protein 1b from Streptococcus pneumoniae

The widespread use of antibiotics has encouraged the development of drug resistance in pathogenic bacteria. In order to overcome this problem, the modification of existing antibiotics and/or the identification of targets for the design of new antibiotics is currently being undertaken. Bifunctional penicillin-binding proteins (PBPs) are membrane-associated molecules whose transpeptidase (TP) activity is irreversibly inhibited by beta-lactam antibiotics and whose glycosyltransferase (GT) activity represents a potential target in the antibacterial fight. In this work, we describe the expression and the biochemical characterization of the soluble extracellular region of Streptococcus pneumoniae PBP1b (PBP1b*). The acylation efficiency for benzylpenicillin and cefotaxime was characterized by stopped-flow fluorometry and a 40-kDa stable TP domain was generated after limited proteolysis. In order to analyze the GT activity of PBP1b*, we developed an electrophoretic assay which monitors the fluorescence signal from PBP1b*-bound dansylated lipid II. This binding was inhibited by the antibiotic moenomycin and was specific for the GT domain, since no signal was observed in the presence of the purified functional TP domain. Binding studies performed with truncated forms of PBP1b* demonstrated that the first conserved motif of the GT domain is not required for the recognition of lipid II, whereas the second motif is necessary for such interaction.     

What controls haploid—diploid ratio in the red alga,Gracilaria verrucosa?

The conditions for maintenance of a haploid—diploid life cycle in the species Gracilaria verrucosa were studied. This species is a red alga, where haploid plants have separate sexes. In the two natural populations studied, male and female haploid individuals were in equal proportions, and the frequency of diploid individuals reached 0.5. A two-fold advantage in viability for diploid relative to haploid juveniles was observed in the field. This advantage can account for a frequency of 0.5 of diploid individuals in natural populations. Different types of anomalies in the reproduction of diploid individuals were observed, all of which lead to a reduction of the haploid stage.