Genetic organization of the BCAM1871 locus.

<p>BCAM1871 is located downstream from <i>cepI</i> with 52 bp separating <i>cepI</i> and BCAM1871 which were determined to be co-transcribed (solid line) using RT-PCR. Size (bp) of each ORF is indicated. The <i>cepI</i> transcription start site is located 28 bp upstream of the ATG start codon <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037611#pone.0037611-Weingart1" target="_blank">[18]</a>. One promoter (arrow) was identified for the <i>cepI</i>-BCAM1871 operon, and this was located upstream of <i>cepI</i> and included in the <i>cepI</i> promoter::<i>lux</i> fusion (pCP300). Nomenclature used to describe K56-dI2 and K56-2ΔBCAM1871 mutants.</p

Bacterial persistence and inflammation in a rat chronic respiratory infection model.

<p>At seven days postinfection rat lungs were harvested and used for (A) quantitative bacteriology by plating lung homogenates and determining the number of colony forming units or (B) quantitative histopathology analysis of hematoxylin and eosin stained lung sections. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037611#s2" target="_blank">Results</a> are displayed using scatter plots with mean values represented by the horizontal bars. P values indicate significant differences in lungs infected with K56-2ΔM1871 compared to that in K56-2.</p

Effects of BCAM1871 on transcription, phenotype and virulence of <i>B. cenocepacia</i>.

<p>BCAM1871 positively (+) influences transcription of several genes invoved in altering phenotypes that contribute to pathogenesis in rat and nematode infection models.</p

Transcription of <i>shvR</i> and <i>afcA</i>.

<p>Transcription was monitored using promoter::<i>lux</i> fusions in LB at 37°C. (A) <i>shvR</i> expression was significantly decreased in K56-2ΔM1871 compared to that in K56-2 from 7–13 h and 28.5–38.5 h (p<0.001). (B) <i>afcA</i> expression was significantly decreased in K56-2ΔM1871 compared to that in K56-2 from 26–44 h (p<0.001).</p

Transcription of <i>cepIR</i> and <i>cciIR</i>.

<p>Transcription was monitored using promoter::<i>lux</i> fusions in LB ± OHL at 37°C. OHL was added at 8 h (arrow). (A) <i>cepI</i> (pCP300) expression was: significantly decreased in K56-2ΔM1871 compared to that in K56-2 from 17.5–28.5 h (p<0.05); significantly increased in K56-2ΔM1871 300 ρM OHL compared to that in K56-2 from 19.5–30 h p<0.001 and 37.5–44 h (p<0.01). (B) <i>cepR</i> (pRM432) expression was: significantly decreased in K56-2ΔM1871 compared to that in K56-2 from 4–12.5 h (p<0.001); significantly increased in K56-2ΔM1871 30 ρM OHL compared to that in K56-2 from 11.5–22.5 h (p<0.01); significantly increased in K56-2ΔM1871 300 ρM OHL compared to that in K56-2 from 8.5–25 h (p<0.001). (C) <i>cciIR</i> (pRM445) expression was: significantly decreased in K56-2ΔM1871 compared to that in K56-2 from 22.5–48 h (p<0.001).</p

Nematode survival and transcription of <i>aidA</i>.

<p>(A) Nematode survival was monitored by analyzing response to touch after feeding on strains indicated. *, significantly decreased in K56-2ΔM1871 (pUCP28T) or K56-dI2 (pUCP28T) compared to that in K56-2 (pUCP28T) (p<0.0001). §,significantly increased in K56-2ΔM1871 (p28T-M1871) compared to that in K56-2ΔM1871 (pUCP28T) (p<0.0001). Values are the means ± standard error of results from plates containing at least 25 worms per plate and are representative of results from at least two individual trials. (B) Transcription was monitored using promoter::<i>lux</i> fusions in LB ± OHL at 37°C. OHL was added at 8 h (arrow). <i>aidA</i> expression was: significantly decreased in K56-2ΔM1871 compared to that in K56-2 from 29.5–42.5 h (p<0.05) (most timepoints); significantly increased in K56-2ΔM1871 300 ρM OHL compared to that in K56-2 from 30–41 h (p<0.001).</p

Protease activity and transcription of <i>zmpA</i> and <i>zmpB</i>.

<p>Protease activity was determined using D-BHI with 1.5% skim milk agar plates after 40 h incubation at 37°C, ±2500 ρM OHL. (A) Significantly different compared to: a, K56-2; b, K56-dI2. (B) Significantly different compared to: a, K56-2 (pUCP28T); b, appropriate parent strain K56-2ΔM1871 (pUCP28T) or K56-dI2 (pUCP28T). All p values <0.001. Transcription was monitored using promoter::<i>lux</i> fusions in LB ± OHL at 37°C. OHL was added at 8 h (arrow). (C) <i>zmpA</i> (pBS13) expression was: significantly decreased in K56-2ΔM1871 compared to that in K56-2 from 14–14.5, 16–22 and 27.5–37.5 h (p<0.05). (D) <i>zmpB</i> (pBS9) expression was: significantly decreased in K56-2ΔM1871 compared to that in K56-2 from 11.5–48 h (p<0.05).</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Influence of BCAM1871 on AHL activity.

<p>AHL activity was monitored using <i>A. tumefaciens</i> A136 (pCF218) (pMV26) in a real-time liquid co-culture assay. (A) Significantly decreased in K56-2ΔM1871 (pUCP28T) compared to that in K56-2 (pUCP28T) from 16–30 h. Significantly increased in K56-2ΔM1871 (p28T-M1871) compared to that in K56-2ΔM1871 (pUCP28T) from 18–30 h. Significantly decreased in K56-dI2 (pUCP28T) compared to that in K56-2ΔM1871 (pUCP28T) from 16–30 h. (B) Significantly increased in K56-2 (p28T-M1871) compared to that in K56-2 (pUCP28T) from 2–10 h and 18–30 h. Significantly increased in K56-2 (pSLS250) compared to that in K56-2 (p28T-M1871) from 4–25 h. (C) Significantly increased in K56-2ΔM1871 (pSLS250) compared to that in K56-2ΔM1871 (pUCP28T) from 2–30 h. No significant difference in K56-dI2 (pUCP28T) compared to that in K56-dI2 (p28T-M1871). All p values<0.001.</p

Oligonucleotide primers used in this study.

a<p>Restriction enzyme sites underlined.</p