Reducing phage production does not rescue the biofilm formation defect of lysogenized strains. (A) Phage titer of LysNeg6 containing the pDMS3Early construct and the empty pMQ78 vector was assayed as described in Materials and Methods. (B) Biofilm formation of WT and LysNeg6 containing the pDMS3Early construct and the pMQ78 vector control was assayed as described in Materials and Methods. OD₅₅₀, optical density at 550 nm.

Quantifying growth and phage production. (A) Planktonic growth of the WT Pseudomonas aeruginosa PA14 (triangles), a biofilm-negative lysogenized strain (LysNeg6; squares), and a biofilm-positive transposon mutant derivative of LysNeg6 (LysNeg6 33310::TnM mutant; diamonds). (B) Phage production in PFU/well of a biofilm-negative lysogenized strain (LysNeg6; squares) and a biofilm-positive transposon mutant derivative of LysNeg6 (LysNeg6 33310::TnM mutant; diamonds). Phage was collected from the same growth assay shown in panel A.

qRT-PCR analysis of the PA14_33310 gene in DMS3 lysogens. (A) qRT-PCR was performed as described in Materials and Methods, and expression was normalized to the rplU gene. Levels of expression of the cas gene PA14_33310 were similar for WT P. aeruginosa PA14 and two DMS3-lysogenized strains which are inhibited for biofilm formation and swarming, LysNeg8 and LysNeg6. However, no expression was detected for two biofilm- and swarming-positive lysogenized strains, LysPos6-3 and LysPos5. Comparable results were obtained when expression of two other cas genes, PA14_33300 and PA14_33320, was assessed (not shown). (B) qRT-PCR was performed on the WT strain grown planktonically or attached to a surface. Shown is the relative expression for the PA14_33310 gene. The expression of the PA14_33310 gene under planktonic conditions is arbitrarily set at 1. ns, not significant (P > 0.05).

CRISPR region of P. aeruginosa PA14. Shown in the center of the figure is the chromosomal organization of the CRISPR genomic region, including the two CRISPRs and the CRISPR-associated (cas) genes. The PA14 gene designation is listed above each gene, and the corresponding homolog from Y. pestis is shown below each gene. The sizes of the ORFs are as follows: for PA14_33300, 504 bp; for PA14_33310, 1,029 bp; for PA14_33320, 984 bp; for PA14_33330, 1,305 bp; for PA14_33340, 3,231 bp; and for PA14_33350, 975 bp. CRISPR-2 is annotated as PA14_33360 in the PA14 genome and is 1,395 bp. The organization of the CRISPRs is also shown. Black boxes indicate repeats, and gray boxes indicate spacers containing identical or partial nucleotide sequence matches from the indicated bacteriophages. The appropriate spacer number is listed above each spacer with known sequence identity to a phage, as is the last spacer number, which indicates the total number of spacers in each CRISPR.

Biofilm and swarming phenotypes of P. aeruginosa PA14 and two DMS3-lysogenized strains. (A) Biofilm formation assessed by crystal violent staining of biofilms grown in microtiter wells for 8 h. (B) Quantification of staining of wells in panel A after solubilization with acetic acid and measurement of optical density at 550 nm (OD₅₅₀). (C) CFU per well of biofilm bacteria. (D) Phase-contrast micrographs of air-liquid interface of biofilms grown in microtiter wells. Experiments displayed in panels A to D were conducted in a polyvinylchloride microtiter plate assay in M63 minimal medium supplemented with arginine (0.4%) and MgSO₄ (1 mM); incubation was at 37°C for 3 h. Bars, 20 μm. (E) Photographs of representative swarming assays after ∼16 h of growth.

Map of the DMS3 genome. The arrows represent the predicted phage DMS3 ORFs and the predicted direction of gene transcription, with the ORF number appearing in bold above the arrow (see Table S2 in the supplemental material for more details regarding each ORF). Black arrows represent genes that were at least partially detected by both GeneMark.hmm and BLASTX, while gray arrows represent genes that were detected only by GeneMark.hmm. White areas represent intergenic regions. The organisms and phages included in the similarity profile were selected for having matches based on BLASTP expect values of less than 1e-19. The bacteriophage genomic sequences in the alignment and the abbreviation for each organism can be found in the lower right of the figure. The boxes labeled other (OT) represent similarity with expect values of less than 1e-19 for an organism that is not one of the 13 listed. The shaded boxes under each gene represent different degrees of similarity to detected genes based on the BLASTP expect values (the key is in the lower right of the figure). The “#” symbol represents the organism with the lowest expect value from BLASTP when the corresponding DMS3 ORF was analyzed. The “&” symbol indicates the organism most closely related to the DMS3 ORF by phylogenic analysis. The genes contained in each transcriptional unit are listed in the upper right of the figure.

Biofilm and swarming phenotypes of PA14_33310 mutants and their response to DMS3 lysogeny. (A) Photographs of representative swarming assays after 24 h of growth of WT Pseudomonas aeruginosa PA14, a biofilm-negative lysogenized strain (LysNeg6), a mutant with a clean deletion of PA14_33310 (Δ33310), and the clean deletion of PA14_33310 expressing the PA14_33310 from a plasmid (Δ33310 + pMQ70-33310). DMS3 lysogens of these mutants are noted with the prefix Lys. (B) Biofilm formation assessed by crystal violet staining of biofilms grown in polyvinylchloride microtiter wells. (C) Quantification of staining of microtiter wells after solubilization with acetic acid and measurement of optical density at 550 nm (OD₅₅₀). Experiments displayed in panels B and C were conducted in a polyvinylchloride microtiter plate assay in M63 minimal medium supplemented with arginine (0.4%) and MgSO₄ (1 mM); incubation was at 37°C for 24 h. Similar results were obtained for biofilms assayed at 8 h (not shown). The empty vector pMQ70 had no effect on any of the phenotypes shown (data not shown).