## 3 Results  
### Hfq Deletion Results in Bacteria with Faster Growth and More Active Metabolism  
Since Hfq is a global regulator that impacts growth and metabolism in bacteria [22; 23], we first analyzed these processes in _Y. ruckeri_ cultures. For this purpose, a mutant strain deficient in this regulatory protein was constructed (\(\Delta\)_Hfq_ strain) prior to analyzing the growth curves, replication profiles and metabolic status. We observed that _Y. ruckeri_\(\Delta\)_Hfq_ exhibited an accelerated bacterial growth in comparison with the wild-type strain (Figure 1a). The bacterial replication in the culture medium was accurately determined by a fluorescence dilution (FD) assay based on a replication reporter system, as previously described [18]. The FD results supported the analysis of growth curves, showing that the \(\Delta\)_Hfq_ strain replicates faster than the wild type, displaying a more accelerated profile of red fluorescence dilution as compared to the wild-type strain (Figure 1b). In accordance with the growth and replication phenotypes, the intracellular levels of ATP from stationary cultures of the \(\Delta\)_Hfq_ strain were higher than that of the wild-type (Figure 1c). In agreement with a more active metabolic state, _Y. ruckeri_\(\Delta\)_Hfq_ also showed a lower NAD\({}^{+}\)/NADH ratio than the wild-type (Figure 1d), evidencing a more reductive intracellular environment. The pHfq complemented strain showed phenotypes comparable to the wild-type strain. All these results suggest that Hfq modulates bacterial proliferation and energy metabolism. Y. ruckeri \(\Delta\)lifq Exhibits Increased Susceptibility to Sublethal Concentrations of Different Antibiotics  
To gain better insights into the role of Hfq in antibiotic response in _Y. ruckeri_, we first evaluated the susceptibilities of the \(\Delta\)lifq and wild-type strains by determining the minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) with different bactericidal antibiotics. The deletion of _lifq_ increased the susceptibility 10-fold compared to all the tested antibiotics, except for florfenicol (Table 2). Then, the strains were grown in the presence of sublethal concentrations of the antibiotics, confirming that the \(\Delta\)lifq strain presented enhanced susceptibility toward most of the antibiotics tested, except for florfenicol (Figure 2a-e). The highest susceptibility was observed for polymysxin B, suggesting that Hfq is critical for regulating outer membrane homeostasis. Overall, these results revealed that the efficiency of the antibiotics on the bacterial growth was higher with the rapidly dividing \(\Delta\)lifq cells having high metabolic rates. \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multirow{2}{*}{**Antibiotic**} & \multicolumn{3}{c}{**MIC (mg mL\({}^{-1}\))**} & \multicolumn{3}{c}{**MBC (mg mL\({}^{-1}\))**} \\ \cline{2-7}  & **WT** & \(\Delta\)_lifq_ & **pHq** & **WT** & \(\Delta\)_lifq_ & **pHq** \\ \hline Ampicillin & 1 & 0.1 & 1 & 1 & 0.1 & 1 \\ Oxolinite acid & 0.005 & 0.0005 & 0.005 & 0.05 & 0.5 & 0.05 \\ Ciprofloxacinacin & 0.003 & 0.003 & 0.003 & 0.003 & 0.3 & 0.003 \\ Florfenicol & 0.01 & 0.01 & 0.01 & 0.01 & 0.1 & 0.01 \\ Polymysxin B & 2 & 0.2 & 2 & 2 & 0.2 & 2 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: MIC and MBC measurements for wild-type (WT), \(\Delta\)lifq and pHfq (complemented) strains in TSB medium supplemented with antibiotics. Figure 1: Growth curves, bacterial replication and metabolic status of _Y. ruckeri_ strains. (**a**) Wild-type (WT), \(\Delta\)lifq and pHfq (complemented) strains were grown in TSB medium and OD\({}_{600}\) was measured at different time points. Data represent the means \(\pm\) standard deviations (\(n=3\)). (**b**) Flow cytometric detection of DsRed and EGFP fluorescence at different time points from the WT, \(\Delta\)lifq and pHfq strains (carrying pDiGc plasmid) cultured in TSB medium for 12 h (\(n=30000\) events analyzed at each time point).