A genome-wide approach for identification and characterisation of metabolite-inducible systems.

Inducible gene expression systems are vital tools for the advancement of synthetic biology. Their application as genetically encoded biosensors has the potential to contribute to diagnostics and to revolutionise the field of microbial cell factory development. Currently, the number of compounds of biological interest by far exceeds the number of available biosensors. Here, we address this limitation by developing a generic genome-wide approach to identify transcription factor-based inducible gene expression systems. We construct and validate 15 functional biosensors, provide a characterisation workflow to facilitate forward engineering efforts, exemplify their broad-host-range applicability, and demonstrate their utility in enzyme screening. Previously uncharacterised interactions between sensors and compounds of biological relevance are identified by employing the largest reported library of metabolite-responsive biosensors in an automated high-throughput screen. With the rapidly growing genomic data these innovative capabilities offer a platform to vastly increase the number of biologically detectable molecules.

Experimental transmission of avian-like swine H1N1 influenza virus between immunologically naïve and vaccinated pigs.

BACKGROUND: Infection of pigs with swine influenza has been studied experimentally and in the field; however, little information is available on the natural transmission of this virus in pigs. Two studies in an experimental transmission model are presented here, one in immunologically naïve and one in a combination of vaccinated and naïve pigs. OBJECTIVES: To investigate the transmission of a recent 'avian-like' swine H1N1 influenza virus in naive piglets, to assess the antibody response to a commercially available vaccine and to determine the efficiency of transmission in pigs after vaccination. METHODS: Transmission chains were initiated by intranasal challenge of two immunologically naïve pigs. Animals were monitored daily for clinical signs and virus shedding. Pairs of pigs were sequentially co-housed, and once virus was detected in recipients, prior donors were removed. In the vaccination study, piglets were vaccinated and circulating antibody levels were monitored by haemagglutination inhibition assay. To study transmission in vaccinates, a pair of infected immunologically naïve animals was co-housed with vaccinated recipient pigs and further pairs of vaccinates were added sequentially as above. The chain was completed by the addition of naive pigs. RESULTS AND CONCLUSIONS: Transmission of the H1N1 virus was achieved through a chain of six pairs of naïve piglets and through four pairs of vaccinated animals. Transmission occurred with minimal clinical signs and, in vaccinates, at antibody levels higher than previously reported to protect against infection.

Expression of the Arabidopsis Xrn4p 5'-3' exoribonuclease facilitates degradation of tombusvirus RNA and promotes rapid emergence of viral variants in plants.

Rapid RNA virus evolution is a major problem due to the devastating diseases caused by human, animal and plant-pathogenic RNA viruses. A previous genome-wide screen for host factors affecting recombination in Tomato bushy stunt tombusvirus (TBSV), a small monopartite plant virus, identified Xrn1p 5'-3' exoribonuclease of yeast, a model host, whose absence led to increased appearance of recombinants [Serviene, E., Shapka, N., Cheng, C.P., Panavas, T., Phuangrat, B., Baker, J., Nagy, P.D., (2005). Genome-wide screen identifies host genes affecting viral RNA recombination. Proc. Natl. Acad. Sci. U. S. A. 102 (30), 10545-10550]. In this paper, we tested if over-expression of Xrn1p in yeast or expression of the analogous Xrn4p cytoplasmic 5'-3' exoribonuclease, which has similar function in RNA degradation in Arabidopsis as Xrn1p in yeast, in Nicotiana benthamiana could affect the accumulation of tombusvirus RNA. We show that over-expression of Xrn1p led to almost complete degradation of TBSV RNA replicons in yeast, suggesting that Xrn1p is involved in TBSV degradation. Infection of N. benthamiana expressing AtXrn4p with Cucumber necrosis tombusvirus (CNV) led to enhanced viral RNA degradation, suggesting that the yeast and the plant cytoplasmic 5'-3' exoribonuclease play similar roles. We also observed rapid emergence of novel CNV genomic RNA variants formed via deletions of 5' terminal sequences in N. benthamiana expressing AtXrn4p. Three of the newly emerging 5' truncated CNV variants were infectious in N. benthamiana protoplasts, whereas one CNV variant caused novel symptoms and moved systemically in N. benthamiana plants. Altogether, this paper establishes that a single plant gene can contribute to the emergence of novel viral variants.

Intracellularly grown gold nanoparticles as potential surface-enhanced Raman scattering probes.

Gold nanoparticles grown within the intracellular confines of living cells are introduced as potential surface-enhanced Raman scattering (SERS) substrates for confocal Raman spectrometry. Electron microscopy and a silver-enhanced reflectance laser scanning confocal microscopic approach were used to visualize the size, shape, and distribution of intracellularly grown gold nanoparticles (IGAuN) as small as 1 nm. Passive uptake as the conventional approach for delivering nanoparticles inside cells faces the insurmountable challenge of escaping the endosomal/lysosomal pathway. In contrast, IGAuN provides an unprecedented advantage of providing access to cytoplasm and nucleus.

Exploiting alternative subcellular location for replication: tombusvirus replication switches to the endoplasmic reticulum in the absence of peroxisomes.

Plus-strand RNA virus replication takes place on distinct membranous surfaces in infected cells via the assembly of viral replicase complexes involving multiple viral and host proteins. One group of tombusviruses, such as Tomato bushy stunt virus (TBSV), replicate on the surfaces of peroxisomal membranes in plant and yeast cells. Surprisingly, previous genome-wide screen performed in yeast demonstrated that a TBSV replicon RNA replicated as efficiently in yeast defective in peroxisome biogenesis as in the wt yeast (Panavas et al., Proc Natl Acad Sci U S A, 2005). To further test how the lack of peroxisomes could affect tombusvirus replication, we used yeast cells missing either PEX3 or PEX19 genes, which are absolutely essential for peroxisome biogenesis. Confocal microscopy-based approach revealed that the wild-type tombusvirus p33 replication protein accumulated in the endoplasmic reticulum (ER) in pex3Delta or pex19Delta yeast, suggesting that tombusvirus replication could take place on the surface of ER membrane. The activities of the isolated tombusvirus replicase preparations from wt, pex3Delta or pex19Delta yeasts were comparable, demonstrating that the assembly of the replicase was as efficient in the ER as in the authentic subcellular environments. The generation/accumulation of tombusvirus recombinants was similar in wt, pex3Delta and pex19Delta yeasts, suggesting that the rate of mistakes occurring during tombusvirus replication is comparable in the presence or absence of peroxisomes. Overall, this work demonstrates that a tombusvirus, relying on the wt replication proteins, can efficiently replicate on an alternative intracellular membrane. This suggests that RNA viruses might have remarkable flexibility for using various host membranes for their replication.

Restriction analysis of genetic variability of Polish isolates of Tomato black ring virus.

Several different isolates of Tomato black ring virus (TBRV) have been collected in Poland from cucumber, tomato, potato and black locust plants. Biological tests showed some differences in the range of infected plants and the type of symptoms, which was the basis for selection of seven the most biologically different TBRV isolates. According to the sequence of TBRV-MJ, several primer pairs were designed and almost the entire sequence of both genomic RNAs was amplified. The RT-PCR products derived from all tested TBRV isolates were digested by restriction enzymes. On the basis of the restriction patterns, the variable and the conserved regions of the TBRV genome were defined and the relationships between the Polish TBRV isolates established.