Validation of the microarray analysis by RT-PCR and B-galactosidase assay.

(A) Gel electrophoresis of the cDNA amplified with primers within the PA0691, PA0692 and the control gene PA0636 from total RNA of P. aeruginosa cells carrying the plasmids pMMB67EH (empty plasmid) or pMUM3 (overexpressing the vreI ECF sigma factor). Positions of molecular size markers (in base pairs) are indicated. Negative controls containing the same amounts of RNA, primers and inactivated reverse transcriptase, were included in this assay (not shown). (B) P. aeruginosa PAO1 (wild-type) cells containing the lacZ transcriptional fusion pMP0691, and the plasmids pMMB67EH (empty plasmid) (white bars) or pMUM3 (grey bars), were grown in LB with or without 1 mM IPTG. β-galactosidase activity was then measured as described in Materials and Methods.

Presence of antibodies against VreI (PA0675)-regulated proteins in the serum of P. aeruginosa infected patients.

(A) Expression of GST-tagged proteins in E. coli. E. coli DH5 ± cells bearing the indicated GST fusion were grown overnight in LB liquid medium and samples were harvested for total protein preparation as described in Materials and Methods. About 10⁸ cells were loaded on each lane and proteins were separated by 12.5% (w/v) polyacrylamide SDS-PAGE and Coomassie blue stained. Arrows indicate the position of the GST-tagged proteins. The position of the molecular size marker is indicated on the right. (B) Immunodetection of purified GST-tagged proteins using the serum of two different CF patients infected with P. aeruginosa. Western blot reactions were revealed by use of the peroxidase colorimetric method [55].

Analysis of VreI (PA0675) stability and activity.

(A) SDS-PAGE of P. aeruginosa wild-type cells (WT) and the vreR sigma factor regulator mutant bearing the pMUM3RσHA-tag plasmid coding for the VreI-HA-tagged protein. Total proteins (upper panel), and cytosol and membrane fractions (lower panel) were separated in 15% (w/v) acrylamide gel. Log phase cells were incubated 45–60 min with (+ in upper panel, and all samples in lower panel) or without (- in upper panel) 1 mM IPTG. (B) β-galactosidase activity of P. aeruginosa wild-type or PA0676 mutant cells containing the pMP0691bKm plasmid (PA0691::lacZ transcriptional fusion) and the pMMB67EH (empty), the pMUM3 (overexpressing the vreI ECF sigma factor) or the pMMB-PUMA3 (overexpressing the whole PUMA3 CSS system) plasmid. Cells were grown overnight in LB liquid medium in the presence of 1 mM IPTG. The β-galactosidase activity is expressed in Miller Units.

Localization of P. aeruginosa within zebrafish embryos.

(A) Fluorescent images of embryos at 1 dpi with an intermediate dose of P. aeruginosa PAO1/RFP cells overexpressing vreI from the pMUM3 plasmid. These embryos were highly infected and normally died by 24–30 hpi. Embryos in whose PAO1/RFP was not visible at 1 dpi (i.e. embryo at the right side of the first panel) survive by clearing the infection and were indistinguishable from the non-injected group. (B) Fluorescent images of embryos infected with PAO1/RFP (red channel) and subjected to whole mount immunohistochemistry using an anti-acetylated tubulin (AcTub) monoclonal antibody that specifically recognizes the nerves of the embryo (green channel). The last panel shows the red/green overlay. (C) Confocal images of three different focal planes of the embryo shown in (B) with similar color coding. All PAO1/RFP panels clearly show the concentration of P. aeruginosa in and around the spinal cord. In addition single bacteria can be seen in the muscle tissue. No colocalization of neuronal cell bodies or axons with PAO1/RFP was seen. However, a close contact between the axon tracts in the spinal cord and the PAO1/RFP is observed.

Localization of VreA in both wild-type cells and the vreR mutant.
An HA-tagged version of the vreA gene was cloned in the broad-host range vector pMMB67EH under control of the tac promoter and subsequently introduced in mPAO1 wild-type strain and the vreR mutant (PA0676::ISphoA/hah). Cells were grown in LB without IPTG induction, disrupted sonication and both the soluble proteins and washed membrane fractions were isolated. Membrane proteins were loaded in five fold excess as compared to the soluble proteins. Proteins were separated on a 15% SDS-PAGE gels and the VreA-HA protein was visualized using antibodies directed against the HA tag. Molecular weight markers are shown on the left in kDa.