Construction of a novel shuttle vector for use in Haemophilus influenzae and H. parainfluenzae

Haemophilus influenzae is an important human pathogen. A number of complete genome sequences of various haemophili are available; however, functional studies have been limited by the lack of an effective shuttle vector which functions in all strains. Here, we have constructed a shuttle vector, pEJ6, which transfers genes between Escherichia coli and H. influenzae and H. parainfluenzae. The vector contains an origin of replication from pLS88 which is functional in E. coli and H. influenzae. In addition it contains an RP4 mobilisation region. The vector can be introduced by electroporation and conjugation into capsulate and non-typeable H. influenzae and is functional for allelic replacement and mutant complementation. The vector will be useful for investigating gene function in Haemophilus spp.     

miR-122 targets an anti-apoptotic gene, Bcl-w, in human hepatocellular carcinoma cell lines

miR-122, a hepato-specific microRNA (miRNA), is frequently down-regulated in human hepatocellular carcinoma (HCC). In an effort to identify novel miR-122 targets, we performed an in silico analysis and detected a putative binding site in the 3′-untranslated region (3′-UTR) of Bcl-w, an anti-apoptotic Bcl-2 family member. In the HCC-derived cell lines, Hep3B and HepG2, we confirmed that miR-122 modulates Bcl-w expression by directly targeting binding site within the 3′-UTR. The cellular mRNA and protein levels of Bcl-w were repressed by elevated levels of miR-122, which subsequently led to reduction of cell viability and activation of caspase-3. Thus, Bcl-w is a direct target of miR-122 that functions as an endogenous apoptosis regulator in these HCC-derived cell lines.     

Control of Anthocyanin Synthesis in PETUNIA HYBRIDA by Multiple Allelic Series of the Genes An1 and An2

A mutable allele of the An1 locus in Petunia hybrida has given rise to a multiple series of stable derivative alleles. Anthocyanin concentration in mature flowers of these mutants (an1^{+/ p}/an1) decreases from the wild-type red to the recessive white in a continuous series. Anthocyanin composition changes regularly: the ratio of peonidin to cyanidin is 3.5 for an an1^+/+/ an1 and 1.2 for an an1^+/p5/an1 mutant. Analysis of anthocyanins during flower development indicates that these differences in composition are due to the specific state of the An1 locus and not to anthocyanin concentration. Anthocyanin concentration in flowers of the allelic series for An1 correlates with the activity of the enzymes UDP-glucose: flavonoid-3-O-glucosyltransferase and SAM: anthocyanin-3'- O-methyltransferase. The same correlations were found for members of a comparable allelic series at the An2 locus. The possibility that the correlation between the enzyme activities is due to the occurrence of a multienzyme complex is discussed.     

Costs and benefits of Wolbachia infection in immature Aedes albopictus depend upon sex and competition level

Bacterial endosymbionts induce various effects on hosts and can dramatically impact host fitness and development. An example is provided by obligate, maternally-inherited Wolbachia, which infect a broad range of invertebrates. Wolbachia are capable of altering host reproduction, thereby promoting infection spread. Wolbachia also pose direct physiological costs and benefits to hosts, complicating their categorization as parasites or mutualists. This study examines for an effect of Wolbachia infection in intra-specific larval competition by Aedes albopictus mosquitoes, with the goal of examining for an impact of Wolbachia infection in mixed populations. Similar to prior work examining for an influence of Wolbachia infection on the fitness of A. albopictus in adults, the results presented here support the hypothesized impact of Wolbachia across all life stages, including immatures. The differential competitiveness of infected larvae detected in our experiments indicates that Wolbachia infected A. albopictus females are less competitive relative to uninfected females when competing under highly competitive conditions. In contrast, under low competitive pressures, infected females experience higher survivorship. Thus, Wolbachia infection shifts from parasitism to mutualism as a function of developmental conditions. Results are discussed in relation to the invasion and persistence of Wolbachia in A. albopictus populations.The results are important to the evolution of stable Wolbachia symbioses, including Wolbachia invasion of an uninfected population. The resulting infection dynamics that occur in an infected population are discussed.     

The Stenopodainae (Hemiptera,Heteroptera) of Argentina

In Argentina, 10 genera and 33 species of Stenopodainae (Hemiptera: Reduviidae) have been recorded. Diagnoses of the genera, subgenera and species are given, and an illustrated key to genera is provided. Six species are new records for Argentina and an additional seven species represent new records for provinces.     

Self-organization at the origin of life

The concept of an (M,R) system with organizational invariance allows one to understand how a system may be able to maintain itself indefinitely if it is coupled to an external source of energy and materials. However, although this constitutes an important step towards understanding the difference between a living and a non-living system, it is not clear that an (M,R) system with organizational invariance is sufficient to define a living system. To take a further step towards defining what it means to be alive it is necessary to add to a simple (M,R) system some property that represents its identity, and which can be maintained and modified in subsequent generations.     

Sequence and expression of an isocitrate dehydrogenase-encoding gene from a polycyclic aromatic hydrocarbon oxidizer,Sphingomonas yanoikuyae B1

An 18.5-kb DNA fragment was cloned from Sphingomonas yanoikuyae (Sy) B1 (previously Beijerinckia B1). Analysis of a 4.3-kb sequence revealed an isocitrate dehydrogenase (Idh)-encoding gene (idhA), an unidentified open reading frame (ORF) and a partial glucosamine synthetase-encoding ORF (glmS). As in a number of bacteria, Tn7 insertion was found specifically at a site past the stop codon of glmS. The predicted 406-amino-acid sequence of IdhA shows, for the first time, an extensive sequence identity (66%) with an eukaryotic NADP⁺-specific Idh. The idhA gene was expressed in Escherichia coli. Identical restriction fragments carrying idhA were found in B1, Sy IFO15102 and Sy Q1 (formerly S. paucimobilis Q1), indicating a well-conserved idh gene.     

AtDSEL, an Arabidopsis cytosolic DAD1-like acylhydrolase, is involved in negative regulation of storage oil mobilization during seedling establishment

Mobilization of seed storage reserves is essential for seed germination and seedling establishment. Here, we report that AtDSEL, an Arabidopsis thalianaDAD1-like Seedling Establishment-related Lipase, is involved in the mobilization of storage oils for early seedling establishment. AtDSEL is a cytosolic member of the DAD1-like acylhydrolase family encoded by At4g18550. Bacterially expressed AtDSEL preferentially hydrolyzed 1,3-diacylglycerol and 1-monoacylglycerol, suggesting that AtDSEL is an sn-1-specific lipase. AtDSEL-overexpressing transgenic Arabidopsis plants (35S:AtDSEL) were defective in post-germinative seedling growth in medium without an exogenous carbon source. This phenotype was rescued by the addition of sucrose to the growth medium. In contrast, loss-of-function mutant plants (atdsel-1 and atdsel-2) had a mildly fast-growing phenotype regardless of the presence of an exogenous carbon source. Electron microscopy revealed that 5-day-old 35S:AtDSEL cotyledons retained numerous peroxisomes and oil bodies, which were exhausted in wild-type and mutant cotyledons. The impaired seedling establishment of 35S:AtDSEL was not rescued by the addition of an exogenous fatty acid source, and 35S:AtDSEL seedling growth was insensitive to 2,4-dichlorophenoxybutyric acid, indicating that β-oxidation was blocked in AtDSEL-overexpressers. These results suggest that AtDSEL is involved in the negative regulation of seedling establishment by inhibiting the breakdown of storage oils.     

Two Leptinotarsa uridine diphosphate N-acetylglucosamine pyrophosphorylases are specialized for chitin synthesis in larval epidermal cuticle and midgut peritrophic matrix

Uridine diphosphate-N-acetylglucosamine-pyrophosphorylase (UAP) is involved in the biosynthesis of chitin, an essential component of the epidermal cuticle and midgut peritrophic matrix (PM) in insects. In the present paper, two putative LdUAP genes were cloned in Leptinotarsa decemlineata. In vivo bioassay revealed that 20-hydroxyecdysone (20E) and an ecdysteroid agonist halofenozide activated the expression of the two LdUAPs, whereas a decrease in 20E by RNA interference (RNAi) of an ecdysteroidogenesis gene LdSHD and a 20E signaling gene LdFTZ-F1 repressed the expression. Juvenile hormone (JH), a JH analog pyriproxyfen and an increase in JH by RNAi of an allatostatin gene LdAS-C downregulated LdUAP1 but upregulated LdUAP2, whereas a decrease in JH by silencing of a JH biosynthesis gene LdJHAMT had converse effects. Thus, expression of LdUAPs responded to both 20E and JH. Moreover, knockdown of LdUAP1 reduced chitin contents in whole larvae and integument samples, thinned tracheal taenidia, impaired larval–larval molt, larval-pupal ecdysis and adult emergence. In contrast, silencing of LdUAP2 significantly reduced foliage consumption, decreased chitin content in midgut samples, damaged PM, and retarded larval growth. The resulting larvae had lighter fresh weights, smaller body sizes and depleted fat body. As a result, the development was arrested. Combined knockdown of LdUAP1 and LdUAP2 caused an additive negative effect. Our data suggest that LdUAP1 and LdUAP2 have specialized functions in biosynthesizing chitin in the epidermal cuticle and PM respectively in L. decemlineata.     

High-level production of Serratia proteamaculans metalloprotease using a recombinant ABC protein exporter-mediated secretion system in Pseudomonas fluorescens

Serratia proteamaculans metalloprotease (SPP) was successfully secreted by a heterologous ABC protein exporter, the Pseudomonas fluorescens TliDEF, in recombinant host strains. Escherichia coli and P. fluorescens cells containing the SPP-encoding gene showed the extracellular protease activity only when the TliDEF-encoding gene cluster was coexpressed. Recombinant P. fluorescens produced an approximately 34.8-fold higher amount of extracellular SPP than did E. coli. The use of a more nutrient-rich medium and controlled dissolved oxygen conditions was effective in increasing SPP secretion in P. fluorescens batch fermentation (an 8.7-fold increase from 41.8 U/mL to 365.2 U/mL). Therefore, SPP, which could not be secreted without an ABC protein exporter, was produced in large quantities by applying the heterologous TliDEF exporter in P. fluorescens. The results also suggest that the use of the ABC protein exporter in P. fluorescens could be an efficient production platform for an industrially promising type I secretion pathway-dependent enzyme.     

Evidence that N-methyl-N′-nitro-N-nitrosoguanidine induces adaptive response in Bacillus thuringiensis

Bacillus thuringiensis is shown to have an inducible error-free repair system for alkylation damage as found in Escherichia coli and Bacillus subtilis. Growth of cells in the presence of low concentrations of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces an adaptive response which is characterized by an increase in resistance to killing and mutagenesis by challenge with higher concentrations of MNNG. In addition, we have noted with interest that adaptive low doses seem to produce lesions at a rate sufficient to induce an increase of mutation frequency, and inhibition of cell division. The possibility of an interaction between SOS and adaptive responses with these low doses of MNNG is discussed.     

Microbial Degradation of Isopropyl-N-3-Chlorophenylcarbamate and 2-Chloroethyl-N-3-Chlorophenylcarbamate

Microbial degradation of isopropyl-N-3-chlorophenylcarbamate (CIPC) and 2-chloroethyl-N-3-chlorophenylcarbamate (CEPC) was observed in a soil perfusion system. Degradation in perfused soils, and by pure cultures of effective bacterial isolates, was demonstrated by the production of 3-chloroaniline and the subsequent liberation of free chloride ion. Identified isolates effective in degrading and utilizing CIPC as a sole source of carbon included Pseudomonas striata Chester, a Flavobacterium sp., an Agrobacterium sp., and an Achromobacter sp. Identified isolates, effective in degrading and utilizing CEPC as a sole source of carbon, included an Achromobacter sp. and an Arthrobacter sp. CIPC-effective isolates degraded CEPC more slowly than CIPC, whereas CEPC-effective isolates degraded CIPC more rapidly than CEPC. Both CIPC- and CEPC-effective isolates degraded isopropyl N-phenylcarbamate (IPC) more rapidly than either CIPC or CEPC.     

Cannibalism and Predation as Paths for Horizontal Passage of Wolbachia between Terrestrial Isopods

The alpha-proteobacteria Wolbachia are the most widespread endosymbionts in arthropods and nematodes. Mainly maternally inherited, these so-called sex parasites have selected several strategies that increase their vertical dispersion in host populations. However, the lack of congruence between the Wolbachia and their host phylogenies suggests frequent horizontal transfers. One way that could be used for horizontal Wolbachia transfers between individuals is predation. The aim of this study was to test whether horizontal passage of Wolbachia is possible when an uninfected terrestrial isopod eats an infected one. After having eaten Armadillidium vulgare harbouring Wolbachia, the predator-recipients (the two woodlice A. vulgare and Porcellio dilatatus dilatatus) that were initially Wolbachia-free were tested positive for the presence of Wolbachia both by quantitative PCR and Fluorescence in situ Hybridization (FISH). Even if the titers were low compared to vertically infected individuals, this constitutes the first demonstration of Wolbachia occurrence in various organs of an initially uninfected host after eating an infected one.     

The mechanical role of VASP in an Arp2/3-complex-based motility assay

The comet motility assay, inspired by Listeria locomotion, has been used extensively as an in vitro model to study the structural and motile properties of the actin cytoskeleton. However, there are no quantitative measurements of the mechanical properties of these actin comets. In this work, we use nanoindentation based on atomic force microscopy to measure the elastic modulus of actin comets grown on  1-μm-diameter beads in an Arp2/3 (actin-related proteins 2 and 3)-complex-dependent fashion in the absence and in the presence of VASP (vasodilator-stimulated phosphoprotein). Recruitment of VASP to the bead surface had no effect on the initial velocity or morphology of the comets. Instead, we observed an improved contact of the comets with the beads and an increased elastic modulus of the comets. The VASP-mediated increase in elastic modulus was dependent on both concentration and ionic strength. In conclusion, we propose that VASP plays a mechanical role in Arp2/3-complex-dependent motility by amplifying the elastic modulus of the thus assembled actin network and, consequently, by strengthening its cohesion for persistent protrusion.     

Leishmania infection and host-blood feeding preferences of phlebotomine sandflies and canine leishmaniasis in an endemic European area,the Algarve Region in Portugal

The Algarve Region (AR) in southern Portugal, which is an international tourist destination, has been considered an endemic region of zoonotic leishmaniasis caused by Leishmania infantum since the 1980s. In the present study, phlebotomine and canine surveys were conducted to identify sandfly blood meal sources and to update the occurrence of Leishmania infection in vectors and dogs. Four sandfly species were captured: Phlebotomus perniciosus, Phlebotomus ariasi, Phlebotomus sergenti and Sergentomyia minuta. In one P. perniciosus female, L. infantum DNA was detected. Blood meal tests showed that this species had no host preferences and was an opportunistic feeder. An overall canine leishmaniasis (CanL) seroprevalence of 16.06% was found; the seroprevalence was 3.88% in dogs housed in kennels and 40.63% in dogs that attended veterinary clinics. The simultaneous occurrence of dogs and P. perniciosus infected with L. infantum in the AR indicates that the region continues to be an endemic area for CanL. Our results reinforce the need for the systematic spatial distribution of phlebotomine populations and their Leishmania infection rates and the need to simultaneously perform pathogen monitoring in both invertebrate and vertebrate hosts to investigate the transmission, distribution and spreading of Leishmania infection.     

One-third rules with equality: Second-order evolutionary stability conditions in finite populations

The one-third law of evolutionary dynamics [Nowak et al. 2004. Emergence of cooperation and evolutionary stability in finite populations. Nature 428, 246-650] describes a robustness criterion for evolution in a finite population: If at an A-frequency of 1/3, the fitness of an A player is greater (smaller) than the fitness of a B player, then a single A mutant that appears in a population of otherwise all B has a fixation probability greater (smaller) than the neutral threshold 1/N, the inverse population size. We examine the case where at an A-frequency of 1/3, the fitness of an A player is exactly equal to the fitness of a B player. We find that in this case the relative magnitude of the cross payoffs matters: If the payoff of A against B is larger (smaller) than the payoff of B against A, then a single A mutant has a fixation probability larger (smaller) than 1/N. If the cross payoffs coincide, we are in the special case of a partnership game, where the deviation cost from an inefficient equilibrium is exactly balanced by the potential gain of switching to the payoff dominant equilibrium. We show that in this case the fixation probability of A is lower than 1/N. Finally, we illustrate our findings by a language game with differentiated costs of signals.     

A bifunctional exoglucanase-endoglucanase fusion protein

A fusion was constructed between the cex gene of Cellulomonas fimi, which encodes an exoglucanase, and the cenA gene of the same organism, which encodes an endoglucanase. The cex-cenA fusion was expressed in Escherichia coli to give a fusion protein with both exoglucanase and endoglucanase activities. The fusion protein, unlike the cex and the cenA gene products from E. coli, did not bind to microcrystalline cellulose, presumably because it lacked an intact substrate-binding region. The fusion protein was exported to the periplasm in E. coli.     

Taxonomy of the hyper-diverse ant genus Tetramorium Mayr in the Malagasy region (Hymenoptera,Formicidae, Myrmicinae) – first record of the T. setigerum species group and additions to the Malagasy species groups with an updated illustrated identification key

In this study we provide an update to the taxonomy of the ant genus Tetramorium Mayr in Madagascar. We report the first record of the Tetramorium setigerum species group in Madagascar and describe the only Malagasy representative as Tetramorium cavernicola sp. n., which is known only from a cave in Ankarana. In addition, we provide an overview of the 19 proposed Malagasy species groups, and discuss their zoogeography and relationships to other groups and larger lineages within the hyper-diverse genus Tetramorium. At present, we recognise a highly unique Malagasy Tetramorium fauna with 113 species endemic to the island of Madagascar out of a total of 125 translating into an endemism rate of 93%. We hypothesise that this fauna is based on one or a few colonisation events from the Afrotropical region, with subsequent adaptive radiation in Madagascar. Furthermore, we present an updated and illustrated identification key to the Tetramorium species groups in the Malagasy region.