Ming Xiao

Horizontal transfer of genetic determinants for degradation of phenol between the bacteria living in plant and its rhizosphere.

Phenol and other monocyclic aromatic compounds (MACs) are highly water-soluble and volatile pollutants that plants are unable to completely degrade. Endophytic bacteria with MAC-degrading ability will facilitate phytoremediation, beneficial to plant survival in contaminated soil. Endophytic bacteria, strains FX1–FX3, and rhizosphere bacteria, strains FX0, FX4, and FX5, were isolated from the root tissue of a corn plant (Zea mays) and the corn rhizosphere near a chemical plant, respectively. The strains FX1–FX5 were able to grow on phenol and reduce phenol concentration, but the strain FX0 was unable to. The strains FX1, FX3, and FX4 were classified as Pseudomonas fluorescens and FX0, FX2, and FX5 as Burkholderia cepacia. The plasmids isolated from the strains FX1–FX5 were found to possess similar traits and to be loaded with a gene encoding the catechol 2, 3-dioxygenase (C23O), a key enzyme in the phenol degradation pathway. Alignment and phylogenetic analysis inferred that in situ horizontal transfer of the C23O gene might have occurred. The horizontal transfer of the C23O gene between endophytic and rhizosphere bacteria was proved by using conjugal matings experiment, in which the transconjugants were found to acquire the plasmid with the C23O gene, able to grow on phenol and degrade phenol.

Influence of NS5A protein of classical swine fever virus (CSFV) on CSFV internal ribosome entry site-dependent translation

An internal ribosome entry site (IRES) present in the 5 untranslated region (UTR) promotes translation of classical swine fever virus (CSFV) genomes. Using an in vitro system with monocistronic reporter RNA containing the CSFV 5UTR, this study found that CSFV NS5A decreased CSFV IRES-mediated translation in a dose-dependent manner. Deletion analysis showed that the region responsible for repressing CSFV IRES activity might cover aa 390-414, located in the C-terminal half of CSFV NS5A. Triple and single alanine-scanning mutagenesis revealed that the inhibitory effect on CSFV IRES-directed translation mapped to the K399, T401, E406 and L413 residues of NS5A. These important amino acids were also found to be present in the NS5A proteins of bovine viral diarrhea virus (BVDV)-1, BVDV-2, border disease virus and hepatitis C virus, indicating that NS5A may play an important role in the switch from translation to replication in these viruses.

In situ degradation of phenol and promotion of plant growth in contaminated environments by a single Pseudomonas aeruginosa strain

For bioremediation of contaminated environments, a bacterial strain, SZH16, was isolated and found to reduce phenol concentration in a selective medium. Using the reaction vessel containing the soil mixed with phenol and bacteria, we found that the single strain degraded efficiently the phenol level in soil samples. The strain was identified as Pseudomonas aeruginosa on the basis of biochemical tests and by comparison of 16S rDNA sequences, and phosphate solubilization and IAA production were not observed in the strain. Simultaneous examination of the role of strain SZH16 in the plant growth and phenol biodegradation was performed. Results showed that inoculation of the single strain in the phenol-spiked soil resulted in corn growth promotion and in situ phenol degradation and the increase in plant biomass correlated with the decrease in phenol content. Colonization experiments showed that the population of the SZH16 strain remained relatively constant. All these findings indicated that the corn growth promotion might be due to reduction in phytotoxicity, a result of phenol biodegradation by the single strain SZH16. Furthermore, the strain was found to stimulate corn growth and reduce phenol concentration simultaneously in phenol-containing water, and even historically contaminated field soils. It is attractive for environment remediation and agronomic applications.

Characterization of NS3, NS5A and NS5B of classical swine fever virus through mutation and complementation analysis.

In order to get further insight into the organization of the pestiviral replication machinery, characterization of NS3, NS5A and NS5B of classical swine fever virus (CSFV) through mutation and complementation analysis was performed. Mutation analysis in genomic replicons and subgenomic replicons indicated importance of the GDD motif in NS5B, the DEYH motif in NS3 and the conserved sequence C2717-C2740-C2742-C2767 in the NS5A for CSFV recover and viral RNA synthesis. Complementation experiments were performed between subgenomic replicons, between RNA replicons or between RNA replicon and expressed nonstructural protein. Rescue of virus and recover of viral RNA synthesis were examined in these complementation experiments. Results showed that mutations within NS5A, neither NS5B nor NS3, can be trans-complemented, strongly suggesting that NS5B and NS3 function in cis mode for regulation of replication. We assumed that the necessary membrane association of CSFV NS5B and NS3 could occur only when they are being translated and originated from an identical translation template, with the exception of NS5A whose membrane association might occur post-translationally.

Classical swine fever virus NS5A regulates viral RNA replication through binding to NS5B and 3'UTR.

In this report, classical swine fever virus (CSFV) NS5A inhibit viral RNA replication when its concentration reached and surpassed the level of NS5B. Three amino acid fragments of CSFV NS5A, 137-172, 224-268 and 390-414 individually were shown to be essential to NS5B binding. The former two fragments were independently necessary for regulation of viral RNA replication and correlated with NS5B and 3UTR binding activity. We also found that amino acids W143, V145, P227, T246, P257, K399, T401, E406 and L413 of CSFV NS5A were essential to NS5B binding activity. Furthermore, these amino acids were shown to be necessary for viral RNA replication and infection and conserved in NS5A proteins of CSFV, BDV, BVDV and HCV. These results indicated that NS5A may regulate viral RNA replication by binding to NS5B and 3UTR. NS5A can still regulate viral RNA synthesis through binding to 3UTR when binding to NS5B is not available.

Classical swine fever virus NS5A protein interacts with 3'-untranslated region and regulates viral RNA synthesis.

To investigate the function of classical swine fever virus (CSFV) NS5A protein, the experiments for viral RNA synthesis and viral replication were performed in the co-presence of NS5A and NS5B. Results showed that small concentrations of NS5A stimulated, large concentrations of NS5A inhibited, viral RNA synthesis and viral replication. Affinity chromatography experiments and UV-crosslinking assays revealed that CSFV NS5A and NS5B bound its cognate 3UTR and that NS5A had higher affinity than NS5B protein in binding to 3UTR. 200 ng of NS5A inhibited NS5B-3UTR complex formation by about 95%. CSFV 3UTR was found to contain two NS5A-binding sites, located in 3UTRSL-1 (nt 161-231) and 3UTRSL-2 (nt 90-160), respectively, a NS5B-binding site, also located in 3UTRSL-1. The 3UTRSL-1 is the common binding site for NS5A and NS5B. Furthermore, competitive electrophoretic mobility shift assays indicated that binding of CSFV NS5A to 3UTRSL-1 is more efficiently than to 3UTRSL-2. These results suggested that the different concentrations of NS5A, the different binding activities of NS5A and NS5B to 3UTR and binding of NS5A to different regions of 3UTR might contribute at least partially to modulation of CSFV replication.

Characterisation of interaction between NS3 and NS5B protein of classical swine fever virus by deletion of terminal sequences of NS5B

The NS3-NS5B interaction of classical swine fever virus (CSFV) is important for viral replication. For characterisation of the interaction between the NS3 and NS5B, a series of NS5B mutants with deletion of N-, C-terminal amino acids and quadruple alanine substitution mutations were produced. GST pull-down assays and immunoprecipitation analyses showed that NS5B and some NS5B mutants have NS3 binding activity. Further experimental data indicated that CSFV NS5B might contain two NS3 binding sites, one covering amino acids 63-99 located at the N-terminal end, another covering amino acids 611-642 at the C-terminal end. Assays for RNA-dependent RNA polymerase (RdRp) activity revealed that CSFV NS3 is able to enhance the RdRp activity of NS5B and some NS5B mutants in vitro. The enhancement might be obtained by NS3 binding to the two terminal sequences of NS5B, which could be attractive targets for drug development against CSFV.

Classical swine fever virus NS3 is an IRES-binding protein and increases IRES-dependent translation.

To get more evidences for understanding the role of NS3 in viral translation, we observed the promotive effect of CSFV NS3 on IRES-mediated translation by using dicistronic and monocistronic systems containing the precise segment comprising CSFV IRES. The results for affinity chromatography and UV-crosslinking assays indicated that NS3 bound CSFV IRES and that CSFV NS5A and NS5B could reduce the IRES-NS3 interaction. Further experiments showed that the NS5A also bound the IRES and that NS3 and NS5A bound the same binding sites of the IRES, suggesting that NS3 and NS5A competitively bind the same sites in IRES RNA sequence, thus hampering the interaction CSFV NS3 and IRES. But, CSFV NS5B was not found to interact with the IRES. The inhibitive effect of NS5B on binding of CSFV NS3 to IRES was supposed to result from the NS3-NS5B interaction which has been documented.

Classical swine fever virus NS3 enhances RNA-dependent RNA polymerase activity by binding to NS5B.

NS3 of pestiviruses contains a protease domain and a RNA helicase/NTPase domain. Contradictory results have been reported regarding NS3 in RNA synthesis. To investigate the effect of NS3 on classical swine fever virus (CSFV) NS5B RNA-dependent RNA polymerase activity (RdRp) activity and NS3-NS5B interaction, RdRp reactions, GST-pull-down assays and co-immunoprecipitation analyses containing NS5B and either of NS3 protein and the different truncated NS3 mutants were performed, respectively. We found that NS3 stimulated NS5B RdRp activity in a dose-dependent manner by binding to NS5 through a NS3 protease domain. Furthermore, mapping important regions of the NS3 protease domain was carried out by deletion mutagenesis, associated with RdRp reactions, GST-pull-down assays and co-immunoprecipitation analyses. Results showed that stimulation of CSFV NS5B RdRp activity was obtained by NS3 binding to NS5B through a 31-amino acid fragment at the N-terminal end of NS3 protease domain, which mediated a specific NS3-NS5B interaction.

Influence of the 5'-proximal elements of the 5'-untranslated region of classical swine fever virus on translation and replication.

The 5-terminal sequence spanning nt 1-29 of the 5-untranslated region of classical swine fever virus (CSFV) forms a 5-proximal stem-loop structure known as domain Ia. Deletions and replacement mutations were performed to examine the role of this domain. Deletion of the 5-proximal nucleotides and disruption of the stem-loop structure greatly increased internal ribosome entry site-mediated translation but abolished the replication of the replicons. Internal deletions resulting in a change in the size of the loop of domain Ia, and even removal of the entire domain, did not substantially change the translation activity, but reduced the replication of CSFV replicons provided the replicons contained the extreme 5-GUAU terminal sequence. Internal replacements leading to a change in the nucleotide sequence of the loop did not alter the translation and replication activities of the CSFV RNA replicon, and did not influence the rescue of viruses and growth characteristics of new viruses. These results may be important for our understanding of the regulation of translation, replication and encapsidation in CSFV and other positive-sense RNA viruses.