Multiple sequence alignment of Omp3 and Omp7 with known immunogenic OmpAs.

<p>Omp3 was aligned with <i>E. coli</i> OmpA (NP_415477.1) and <i>K. pneumoniae</i> OmpA (ABR76422) while Omp7 was aligned with <i>P. gingivalis</i> OmpAs PG33 (AF175715) and PG32 (AF175714).</p

Putative OmpA genes identified by BLASTP analysis using the OmpA domain sequence (below) as the query against the <i>B. pseudomallei</i> K96243 reference genome sequence.

<p>Conserved OmpA domain sequence used as a query sequence (PF00691):</p><p>LFDFDKATLKPEQQQLLDAIADLLKAIPPDNRVIVEGHTDSRPIGSDEYPSNQALSERRADSVADYLVSKGGVPADRISAVGYGESKPIASNKTEEGRAKNR.</p

Primer sequences used for amplification of the 12 predicted ORFs of <i>Burkholderia pseudomallei</i> OmpA.

<p>Primer sequences used for amplification of the 12 predicted ORFs of <i>Burkholderia pseudomallei</i> OmpA.</p

Screening of OmpA-specific IgG in immunised BALB/c mice.

<p>Mice were immunised with 50 µg of recombinant Omp3 or Omp7. The sera were collected (n = 4) prior to and after immunisation and pooled. The levels of OmpA-specific IgG were analysed by ELISA. The presence of specific antibodies to recombinant Omp3 (closed triangles) and Omp7 (closed squares) proteins was observed in sera from immunised mice but not in pre-immunised sera (open triangles for Omp3 pre-immunised sera and open squares for Omp7 pre-immunised sera) (<i>P</i><0.05). Data represent the mean absorbance (OD_450nm)±SEM.</p

Multiple sequence alignment (C-termini) of predicted amino acid sequences of <i>B. pseudomallei</i> D286 OmpA.

<p>The predicted amino acid sequences of 12 <i>B. pseudomallei</i> OmpAs together with 5 experimentally verified immunogenic OmpAs were aligned. The experimentally verified OmpAs used for the alignment: <i>E. coli</i> OmpA (NP_415477.1), KpmOmpA - <i>K. pneumoniae</i> OmpA (ABR76422), PG33 - <i>P. gingivalis</i> OmpA (AF175715), PG32 - <i>P. gingivalis</i> OmpA (AF175714), RmpM–<i>N. meningitidis</i> OmpA (YP_001599860). The residues implicated for their role in the interactions with peptidoglycans are indicated (asterisks - direct interaction; dots - indirect interaction).</p

Survival of BALB/c mice immunised with recombinant Omp3 or Omp7 and challenged with <i>B. pseudomallei</i>.

<p>Mice were immunised with either Omp3 (open triangles) or Omp7 (open squares) prior to challenge with 1×10⁶ cfu <i>B. pseudomallei</i> by the i.p. route of infection. Both groups immunised with either Omp3 or Omp7 displayed 50% survival rate up to 21 days post-infection. All the control mice (closed squares) received <i>E. coli</i> BL21 (DE3) cells in FIA followed by challenge, and died by day 9 post-challenge.</p

Western blot analysis of purified recombinant OmpA.

<p>Purified recombinant OmpAs were electrophoresed on a 12% SDS-PAGE, transferred onto a nitrocellulose membrane and reacted with (A) monoclonal anti-histidine tag antibody conjugated with HRP. The expected protein sizes for Omp3, 4, 5, 6, 7 and 10 are 27, 27.5, 34, 65, 22.5 and 24.5 kDa respectively. Molecular weight sizes (kDa) of the broad range marker are indicated in the left panel. Among 6 recombinant OmpA proteins, Omp3 and Omp7 reacted strongly with (B) rabbit anti-<i>B. pseudomallei</i> sera and (C) pooled melioidosis patients' sera compared to other Omps. Lane 1; Omp3, lane 2; Omp4, lane 3; Omp5, lane 4; Omp6, lane 5; Omp7, lane 6; Omp10.</p

Analysis of OmpA-specific polyclonal antibody isotypes in immunised BALB/c mice.

<p>Mice were immunised with 50 µg of recombinant Omp3 or Omp7. The sera were collected (n = 4) prior to and after immunisation and pooled. The levels of IgG subclasses and IgM were determined by ELISA. Data represent the mean absorbance (OD_450nm)±SEM. The IgG_2a/IgG₁ ratio of Omp3-immunised mice is 1.2 while for Omp7-immunised mice, the ratio is 1.3. Open bars represent pre-immunised sera, grey bars represent anti-Omp3 antibody and black bars represent anti-Omp7 antibody.</p

Species distribution of annotated <i>Rafflesia cantleyi</i> flower transcripts.

<p>Species distribution of annotated <i>Rafflesia cantleyi</i> flower transcripts.</p

Perigone Lobe Transcriptome Analysis Provides Insights into <i>Rafflesia cantleyi</i> Flower Development

<div><p><i>Rafflesia</i> is a biologically enigmatic species that is very rare in occurrence and possesses an extraordinary morphology. This parasitic plant produces a gigantic flower up to one metre in diameter with no leaves, stem or roots. However, little is known about the floral biology of this species especially at the molecular level. In an effort to address this issue, we have generated and characterised the transcriptome of the <i>Rafflesia cantleyi</i> flower, and performed a comparison with the transcriptome of its floral bud to predict genes that are expressed and regulated during flower development. Approximately 40 million sequencing reads were generated and assembled <i>de novo</i> into 18,053 transcripts with an average length of 641 bp. Of these, more than 79% of the transcripts had significant matches to annotated sequences in the public protein database. A total of 11,756 and 7,891 transcripts were assigned to Gene Ontology categories and clusters of orthologous groups respectively. In addition, 6,019 transcripts could be mapped to 129 pathways in Kyoto Encyclopaedia of Genes and Genomes Pathway database. Digital abundance analysis identified 52 transcripts with very high expression in the flower transcriptome of <i>R</i>. <i>cantleyi</i>. Subsequently, analysis of differential expression between developing flower and the floral bud revealed a set of 105 transcripts with potential role in flower development. Our work presents a deep transcriptome resource analysis for the developing flower of <i>R</i>. <i>cantleyi</i>. Genes potentially involved in the growth and development of the <i>R</i>. <i>cantleyi</i> flower were identified and provide insights into biological processes that occur during flower development.</p></div