RNAs 1 and 2 of the tripartite genome of alfalfa mosaic virus (AMV) encode the replicase proteins P1 and P2, respectively. P1 contains a methyltransferase-like domain in its N-terminal half, which has a putative role in capping the viral RNAs. Six residues in this domain that are highly conserved in the methyltransferase domains of alphavirus-like viruses were mutated individually in AMV P1. None of the mutants was infectious to plants. Mutant RNA 1 was coexpressed with wild-type (wt) RNAs 2 and 3 from transferred DNA vectors in Nicotiana benthamiana by agroinfiltration. Mutation of His-100 or Cys-189 in P1 reduced accumulation of negative-and positive-strand RNA in the infiltrated leaves to virtually undetectable levels. Mutation of Asp-154, Arg-157, Cys-182, or Tyr-266 in P1 reduced negative-strand RNA accumulation to levels ranging from 2 to 38% of those for the wt control, whereas positive-strand RNA accumulation by these mutants was 2% or less. The (transiently) expressed replicases of the six mutants were purified from the agroinfiltrated leaves. Polymerase activities of these preparations in vitro ranged from undetectable to wt levels. The data indicate that, in addition to its putative role in RNA capping, the methyltransferase-like domain of P1 has distinct roles in replication-associated functions required for negative-strand RNA synthesis. The defect in negative-strand RNA synthesis of the His-100 and Cys-189 mutants could be complemented in trans by coexpression of wt P1.

Background: The Burkholderia cepacia complex (Bcc) groups bacterial species with beneficial properties that can improve crop yields or remediate polluted sites but can also lead to dramatic human clinical outcomes among cystic fibrosis (CF) or immuno-compromised individuals. Genome-wide regulatory processes of gene expression could explain parts of this bacterial duality. Transcriptional 70 factors are components of these processes. They allow the reversible binding of the DNA-dependent RNA polymerase to form the holoenzyme that will lead to mRNA synthesis from a DNA promoter region. Bcc genome-wide analyses were performed to investigate the major evolutionary trends taking place in the 70 family of these bacteria.

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The Burkholderia cenocepacia B&B clone was found previously to be responsible for an epidemic outbreak within an intensive care unit in France. This clone belongs to the ST32 clonal complex, which is one of the most prevalent among French cystic fibrosis patients and is known to be related to the highly virulent ET12 clonal complex. Genomic repartition biases of insertion sequences (ISs) were investigated to improve our understanding of the evolutionary events leading to B. cenocepacia diversification and the emergence of such epidemic lineages. IS were used for tracking convergent genetic inactivations and recent DNA acquisitions. B. cenocepacia IS families and subgroups were compared in terms of genetic diversity and genomic architecture using fully sequenced genomes, PCR screening and DNA blot analysis. These analyses revealed several features shared by the B&B and ET12 epidemic clones. IS elements showed a frequent localization on genomic islands (GI) and indicated convergent evolution towards inactivation of certain loci. The IS407 subgroup of the IS3 family was identified as a good indicator of recently acquired GIs in clone ET12. Several IS407 elements showed strain-specific or clonal complex-specific localizations. IS407 DNA probing of a DNA library built from the B. cenocepacia B&B epidemic clone led to the identification of a recently acquired IS407-tagged GI likely to be conjugative and integrative. The B&B clone showed significant differences in its IS architecture from that of ST32 strains isolated from Czech cystic fibrosis patients.