## 2 Materials and Methods  
Bacterial strains and culture conditions: _Y. ruckeri_ strains were routinely grown at 26 \({}^{\circ}\)C in Trypticase soy broth (TSB) or Trypticase soy agar (TSA). The strains and plasmids used in this work are listed in Table 1. pCM433 was donated by [PERSON] (Addgene plasmid # 15670; [[http://n2t.net/addgene:15670](http://n2t.net/addgene:15670)]([http://n2t.net/addgene:15670](http://n2t.net/addgene:15670)), accessed on 29 May 2021, RRID:Addgene_15670), and pDiGe was donated by [PERSON] and [PERSON] (Addgene plasmid # 59322; [[http://n2t.net/addgene:59322](http://n2t.net/addgene:59322)]([http://n2t.net/addgene:59322](http://n2t.net/addgene:59322)), accessed on 29 May 2021; RRID:Addgene_59322). Construction of gene deletions mutants: The deletion mutant strains \(\Delta\)_relA_ and \(\Delta\)_lfq_\(\Delta\)_relA_ (double mutant) were constructed as we previously described for \(\Delta\)_lfq_[18], using an unmarked allelic exchange method based on the sacB-dependent suicide plasmid pCM433 [19]. Briefly, the _relA_ gene sequence of _Y. ruckeri_ CD2 was amplified by PCR with forward and reverse primers containing the _NdeI_ and _BgII_ restriction sites, respectively. Both the PCR product and the pCM433 plasmid were digested with _NdeI_ and _BgII_ and then ligated. The plasmid carrying _relA_ gene was then transformed into _Y. ruckeri_ CD2 to obtain the \(\Delta\)_relA_ strain. Transformants were first selected on TSA plates supplemented with chloramphenicol (0.075 mg mL\({}^{-1}\)) and then plated on TSA supplemented with 5% sucrose for the selection of sucrose-resistance clones. To select for recombinants that have excised the vector, we performed a negative selection on TSA plates supplemented with chloramphenicol (0.075 mg mL\({}^{-1}\)). The \(\Delta\)_relA_ strain was confirmed by PCR, using primers flanking the substitution site. To obtain the double mutant strain \(\Delta\)_relA_\(\Delta\)_lfq_, the pCM433 plasmid carrying the _relA_ gene was transformed into the single \(\Delta\)_lfq_ strain and the selection of the double knock-out mutant was performed as described above. For the complementation assays, _lfq_ was cloned with its promoter into pBR322. The PCR product obtained with primers Hfq_pBR322_EcoRI_Fw and Hfq_pBR322_HindIII_Rv was digested with EcoRI and HindIII and then cloned into pBR322, in the respective restriction sites, to yield pHfq. Specific primers used in this work are listed in Supplementary Table S1. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determination: Bacteria were grown overnight in TSB medium, washed three times with sterile phosphate-buffered saline (PBS), diluted in fresh TSB medium and aliquoted (100 \(\mu\)L) into the wells of sterile microtitre plates (10\({}^{8}\) CFU mL\({}^{-1}\) in each well). MIC and MBC were determined using serial 10-fold microdilution of the antibiotics in TSB medium. The lowest concentration of antibiotics that inhibited growth (measured as the OD\({}_{600}\)) by at least 50%, relative to growth in the absence of antibiotics, was defined as the MIC. MBC was defined as 99.9% killing of the starting inoculum. Persister assay. Persistence was determined by measuring the viable bacteria as colony-forming units (CFU) per mL. The cultures were prepared from different inoculums: exponential phase cells (OD\({}_{600}\) of 0.3), stationary phase cells (OD\({}_{600}\) of 0.9) and bacteria recovered from biofilm. For biofilm assay 100 \(\mu\)L of an overnight culture was inoculated in a 24-well polystyrene microtitre plate containing 900 \(\mu\)L of TSB. The plates were incubated without agitation at 26 \({}^{\circ}\)C for 24 h and then washed three times with PBS. Adherent bacteria were recovered by sonication in PBS and resuspended in fresh TSB. The OD\({}_{600}\) was measured to estimate total cell number for each sample: exponential, stationary and biofilm recovered cells.