64633) and PA14 (accession no. NC_008463, PA14_68470) encode proteins which might be identical
64633) and PA14 (accession no. NC_008463, PA14_68470) encode proteins which are identical to RsmF in IL-1 Antagonist web Strain PAO1. Due to the fact RsmA inhibits P. aeruginosa exopolysaccharide production, rsmA Cathepsin L Inhibitor Storage & Stability mutants generally create robust biofilms (18). None of your mutations, however, led to an altered biofilm phenotype in strain PA103. This was not unexpected for the reason that strain PA103 lacks flagella and features a defect within the Las quorum-sensing program, both of that are expected for robust biofilm formation (191). In contrast, the PA14 rsmA mutant showed a substantial raise in biofilm formation (13-fold) compared with wild form (Fig. 2A). Although the rsmF mutant was indistinguishable from wild type, the PA14 rsmA, rsmF (rsmAF) double mutant created a significantly far more robust biofilm than either wild sort (44-fold increase) or the rsmA mutant (3.5-fold boost). The biofilm phenotype was restored to near wild-type levels within the rsmAF double mutant when either rsmA or rsmF have been supplied in trans. Notably, the PA103 and PA14 rsmA and rsmAF double mutants grew slower than wild form (SI Appendix, Fig. S3 A and B); nevertheless, the modest raise in generation times of your PA14 rsmA and rsmAF mutants (SI Appendix, Fig. S3C) is unlikely to account for their altered biofilm forming capacity. Thesewt activitywt Biofilm1500 1000 500*wt activityA* *B125 125 100 one hundred 75 75 50 50 25 25*C2000 1000 200 100**RsmA Plasmid Strain PApJN105 rsmA rsmF pRsmA pRsmF rsmAFExoU PcrV Plasmid Strain PApJN105 rsmA rsmF pRsmA pRsmF rsmAFHcp1 Tse1 Plasmid Strain PApJN105 rsmA rsmF pRsmA pRsmF rsmAFFig. 2. Contribution of RsmA and RsmF to biofilm formation, T3SS, and T6SS gene expression. (A) The indicated PA14 strains were cultured in glass tubes with shaking for 12 h at 37 in LB medium. Biofilm formation was measured by crystal violet staining and values are reported normalized to percent wild-type (WT) activity (set at 100 ). (B and C) Wild-type PA103 and also the indicated mutants carrying a transcription reporter for (B) T3SS gene expression (PexsD-lacZ) or even a translational reporter for TssA1 translation (PlacUV5-tssA’-‘lacZ) have been transformed with a vector control (pJN105), pRsmA, or pRsmF. Strains were cultured below inducing situations (low Ca2+) for T3SS gene expression inside the presence of 0.4 arabinose to induce rsmA or rsmF expression from the PBAD promoter and assayed for -galactosidase activity. Reported values are in Miller units normalized to percent WT activity (set at 100 ). Statistical variations have been determined employing two-tailed unpaired t tests. *P 0.01. (A ) Whole-cell lysate (A) and culture supernatant fluid (B and C) samples were harvested from an equivalent number of cells and immunoblotted for RsmA (A), or secreted proteins of the T3SS (B; ExoU and PcrV), or T6SS (C; Hcp1 and Tse1).15056 | pnas.org/cgi/doi/10.1073/pnas.Marden et al.results show that although each RsmA and RsmF repress biofilm formation, the contribution of RsmF is only revealed inside the absence of RsmA.Expression of Either rsmA or rsmF in Trans Is Sufficient to Restore T3SS Gene Expression in an rsmAF Mutant. Since RsmA is re-quired for maximal T3SS gene expression (7, 9, 22), we hypothesized that RsmF may perhaps play a related role in controlling T3SS gene expression. To test this hypothesis, we introduced a T3SSdependent reporter gene (PexsD-lacZ) (23) into the ectopic CTX attachment site around the chromosome of wild-type strain PA103 as well as the rsmA, rsmF, and rsmAF mutants. Beneath T3SS-inducing circumstances (low Ca2+.