List of carbon- and nitrogen compounds that were differentially used in micro-oxic versus aerobic conditions.

<p>Biolog plates PM1 and PM2a were used for C-source profiling and plate PM3b for N-sources utilization.</p

Protease activity is increased in micro-oxia.

<p>The exoenzymes cellulase, protease and lipase were measured in supernatants of aerobic (black) and micro-oxic (grey) growing cells as described in material and methods. The activity in the supernatant of aerobic cells was set to 100%. Whiskers indicate SD, n = 6.</p

List of 176 <i>B. cenocepacia</i> H111 genes/proteins that showed differential expression in micro-oxic (M) conditions compared to aerobic (A) conditions (DESeq analysis, p-value<0.15 for proteomics and p-value<0.2 for RNA-Seq).

a<p>Nomenclature and description according to GenBank file CAFQ01000001.1.</p>b<p>Orthologs were identified as described in the Material and Methods section.</p>c<p>Predicted topology (Tp) according to SignalP v4.0 (secreted proteins, S) and TMHMM v2.0 (transmembrane, TM).</p>d<p>Fold change (FC) of protein expression, comparing micro-oxically (M) with aerobically (A) grown wild-type strain.</p>e<p>Fold change (FC) of transcript expression, comparing micro-oxically (M) with aerobically (A) grown wild-type strain.</p><p>nd: The gene was not identified on protein level.</p><p>M only and A only: The gene/protein was detected only micro-oxically (M) or aerobically (A).</p><p>The proNOG categories are indicated and the 58 differentially expressed proteins are indicated in bold. The overlap in low oxygen regulation with strain J2315 (Sass et al., 2013) is indicated in italics.</p

Response of <i>Burkholderia cenocepacia</i> H111 to Micro-Oxia

<div><p><i>B. cenocepacia</i> is an opportunistic human pathogen that is particularly problematic for patients suffering from cystic fibrosis (CF). In the CF lung bacteria grow to high densities within the viscous mucus that is limited in oxygen. <i>Pseudomonas aeruginosa</i>, the dominant pathogen in CF patients, is known to grow and survive under oxygen-limited to anaerobic conditions by using micro-oxic respiration, denitrification and fermentative pathways. In contrast, inspection of the genome sequences of available <i>B. cenocepacia</i> strains suggested that <i>B. cenocepacia</i> is an obligate aerobic and non-fermenting bacterium. In accordance with the bioinformatics analysis we observed that <i>B. cenocepacia</i> H111 is able to grow with as little as 0.1% O₂ but not under strictly anoxic conditions. Phenotypic analyses revealed that H111 produced larger amounts of biofilm, pellicle and proteases under micro-oxic conditions (0.5%–5% O₂, i.e. conditions that mimic those encountered in CF lung infection), and was more resistant to several antibiotics. RNA-Seq and shotgun proteomics analyses of cultures of <i>B. cenocepacia</i> H111 grown under micro-oxic and aerobic conditions showed up-regulation of genes involved in the synthesis of the exopolysaccharide (EPS) cepacian as well as several proteases, two isocitrate lyases and other genes potentially important for life in micro-oxia.</p><p>Data deposition: RNA-Seq raw data files are accessible through the GEO Series accession number GSE48585. MS data have been deposited in the ProteomeXchange database (PXD000270).</p></div

Influence of oxygen on biofilm formation in <i>B. cenocepacia</i> H111.

<p>Biofilm formation in ABC minimal medium. <i>B. cenocepacia</i> H111 was grown in 96-well plates under aerobic (black) or in micro-oxic (grey) conditions created in a CampyGen compact system (oxoid). Whiskers indicate SD, n = 3.</p

Differential protein expression under micro-oxic and aerobic conditions.

<p>MA plot showing the log2 fold change in protein expression of <i>B. cenocepacia</i> H111 grown under micro-oxic versus aerobic conditions. The top regulated proteins are shown in color: proteins with increased expression under micro-oxic conditions are indicated in red, down-regulated proteins in green.</p

The role of the two QS systems in the regulation of selected genes.

<p>The <i>bapA</i> (A), <i>bclA</i> (B), and <i>aidA</i> (C) promoter activities were assessed by means of transcriptional <i>lacZ</i> fusions in the H111 wild type strain and in the mutant defective in AHL and BDSF synthesis (Δ<i>cepI rpfF</i>_Bc). The strains were grown to late exponential growth phase in LB Lennox broth in the absence or presence of signal molecules (200 nM C8-HSL; 10 µM BDSF) as indicated by+and - below each bar. Error bars indicate SEM, n = 3. * P<0.05, ** P<0.01, *** P<0.001 (t-test, two-tailed) compared to Δ<i>cepI rpfF</i>_Bc without signalling molecule (ns, not significant) (D) Expression of BclB and AidA in the H111 wild type and the double mutant Δ<i>cepI rpfF_Bc</i> as assessed by Western Blot analysis. The strains were grown on plates in the presence or absence of signal molecules as indicated by+and - below each band.</p

Schematic presentation of the two <i>B. cenocepacia</i> H111 QS circuitries.

<p>The CepI/CepR system consists of the AHL synthase CepI directing the synthesis of C8-HSL, and of the transcriptional regulator CepR. The RpfF/RpfR system consists of RpfF which directs the synthesis of BDSF, and of its cognate receptor RpfR. Upon binding of BDSF to RpfR the c-di-GMP phosphodiesterase activity of the protein is stimulated and as a consequence the intracellular c-di-GMP level is lowered. The two QS systems operate in parallel to control specific as well as overlapping sets of genes. Our working model assumes an unknown c-di-GMP receptor protein × that stimulates transcription of target genes. Alternatively, the two QS cascades converge and control the expression or the activity status of an unknown common regulator Y, which in turn regulates expression of target genes. C-di-GMP has a negative regulatory effect on AHL levels via an unknown mechanism (depicted by the dashed line).</p

Biofilm formation and protease activity are co-regulated by AHL and BDSF.

<p>(A) Biofilm formation in ABC minimal. (B) Protease activity in NYG medium. The strains tested are the wild type H111 and the <i>cepI rpfF</i>_Bc double mutant. Strains were grown in the presence of absence of signal molecules (200 nM C8-HSL; 10 µM BDSF) as indicated by+and - below each bar. Error bars indicate SEM, n≥3. ** P<0.01, *** P<0.001 (t-test, two-tailed) compared to Δ<i>cepI rpfF</i>_Bc without signalling molecule (ns, not significant).</p

Validation of RNA-Seq results using quantitative PCR analysis.

a<p>Nomenclature according to GenBank file CAFQ01000001.1.</p>b<p>Orthologs were identified as described in the Material and Methods section.</p>c<p>Description according to EggNOG classification.</p>d<p>Fold change (FC) of expression, comparing wild type strain with <i>rpfF</i> mutant grown in LB medium until an OD of 2.</p>e<p>Fold change (FC) of expression, comparing wild type strain with complemented <i>rpfF</i> mutant grown in LB medium until an OD of 2.</p>f<p>Fold change (FC) of expression comparing wild type strain with <i>cepR</i> mutant grown in LB medium until an OD of 2.</p>g<p>Fold change (FC) of expression, comparing wild type strain with complemented <i>cepR</i> mutant grown in LB medium until an OD of 2.</p