Insertion sequence evolutionary patterns highlight convergent genetic inactivations and recent genomic island acquisitions among epidemic Burkholderia cenocepacia.

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.

Epidemiology and molecular characterization of a clone of Burkholderia cenocepacia responsible for nosocomial pulmonary tract infections in a French intensive care unit.

Clustered cases of nosocomial pulmonary infections were observed in a French intensive care unit. Biochemical tests showed the etiologic agents to be part of the Bcc (Bcc). recA polymerase chain reaction-restriction fragment length polymorphism analysis and molecular phylogeny positioned the isolates into Burkholderia cenocepacia. Their recA sequences were found identical to those of ET12 strains responsible of necrotic pneumonia in cystic fibrosis patients. Analyses of a multi locus sequence typing genes set confirmed this proximity and suggested a wide distribution among occidental countries but could not resolve their phylogenetic position unambiguously. A novel marker, ecfB, indicated a significant phylogenetic divergence from ET12 strains. Pulse field gel electrophoresis analysis of SpeI-restricted total genomic DNA of the strains showed a unique profile indicative of a clonal outbreak. Environmental hospital screenings indicated cross-contamination between staff and patients. Bcc strains from outdoor environments were not related to this clone but indicated the presence of Burkholderia multivorans and Burkholderia vietnamiensis.

Architecture of Burkholderia cepacia complex sigma70 gene family: evidence of alternative primary and clade-specific factors, and genomic instability.

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 sigma70 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 sigma70 family of these bacteria. RESULTS: Twenty sigma70 paralogous genes were detected in the Burkholderia cenocepacia strain J2315 (Bcen-J2315) genome, of which 14 were of the ECF (extracytoplasmic function) group. Non-ECF paralogs were related to primary (rpoD), alternative primary, stationary phase (rpoS), flagellin biosynthesis (fliA), and heat shock (rpoH) factors. The number of sigma70 genetic determinants among this genome was of 2,86 per Mb. This number is lower than the one of Pseudomonas aeruginosa, a species found in similar habitats including CF lungs. These two bacterial groups showed strikingly different sigma70 family architectures, with only three ECF paralogs in common (fecI-like, pvdS and algU). Bcen-J2315 sigma70 paralogs showed clade-specific distributions. Some paralogs appeared limited to the ET12 epidemic clone (ecfA2), particular Bcc species (sigI), the Burkholderia genus (ecfJ, ecfF, and sigJ), certain proteobacterial groups (ecfA1, ecfC, ecfD, ecfE, ecfG, ecfL, ecfM and rpoS), or were broadly distributed in the eubacteria (ecfI, ecfK, ecfH, ecfB, and rpoD-, rpoH-, fliA-like genes). Genomic instability of this gene family was driven by chromosomal inversion (ecfA2), recent duplication events (ecfA and RpoD), localized (ecfG) and large scale deletions (sigI, sigJ, ecfC, ecfH, and ecfK), and a phage integration event (ecfE). CONCLUSION: The Bcc sigma70 gene family was found to be under strong selective pressures that could lead to acquisition/deletion, and duplication events modifying its architecture. Comparative analysis of Bcc and Pseudomonas aeruginosa sigma70 gene families revealed distinct evolutionary strategies, with the Bcc having selected several alternative primary factors, something not recorded among P. aeruginosa and only previously reported to occur among the actinobacteria.

Selection of nitrogen-fixing deficient Burkholderia vietnamiensis strains by cystic fibrosis patients: involvement of nif gene deletions and auxotrophic mutations.

Burkholderia vietnamiensis is the third most prevalent species of the Burkholderia cepacia complex (Bcc) found in cystic fibrosis (CF) patients. Its ability at fixing nitrogen makes it one of the main Bcc species showing strong filiations with environmental reservoirs. In this study, 83% (29 over 35) of the B. vietnamiensis CF isolates and 100% of the environmental ones (over 29) were found expressing the dinitrogenase complex (encoded by the nif cluster) which is essential in N(2) fixation. Among the deficient strains, two were found growing with ammonium chloride suggesting that they were defective in N(2) fixation, and four with amino acids supplements suggesting that they were harbouring auxotrophic mutations. To get insights about the genetic events that led to the emergence of the N(2)-fixing defective strains, a genetic analysis of B. vietnamiensis nitrogen-fixing property was undertaken. A 40-kb-long nif cluster and nif regulatory genes were identified within the B. vietnamiensis strain G4 genome sequence, and analysed. Transposon mutagenesis and nifH genetic marker exchanges showed the nif cluster and several other genes like gltB (encoding a subunit of the glutamate synthase) to play a key role in B. vietnamiensis ability at growing in nitrogen-free media. nif cluster DNA probings of restricted genomic DNA blots showed a full deletion of the nif cluster for one of the N(2)-fixing defective strain while the other one showed a genetic organization similar to the one of the G4 strain. For 17% of B. vietnamiensis clinical strains, CF lungs appeared to have favoured the selection of mutations or deletions leading to N(2)-fixing deficiencies.

Role of the alfalfa mosaic virus methyltransferase-like domain in negative-strand RNA synthesis.

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.