## 2 Results  
### rsh Deletion Drastically Impacts Growth in Minimal Medium and the Infection Process  
We generated a \(\Delta\)_rsh_ strain by allelic replacement in _B. abortus_ 544 and we assayed the growth of this strain in rich culture medium (2 YT) as well as in Plommet minimal medium [23] supplemented with erythroid as a carbon source. The growth of \(\Delta\)_rsh_ in 2 YT was similar to the WT strain during the exponential phase, but the shift into the stationary phase occurred later and at a higher optical density (OD) compared to the wild type strain (Figure 1A). To evaluate bacterial viability, we counted the colony forming units (CFUs) throughout the culture in liquid rich medium (Figure 2). The \(\Delta\)_rsh_ strain showed a clear survival defect during the stationary phase, marked by a decrease in CFUs between 24 h and 48 h compared to the WT and the complemented strain.  
Because it has been shown in other bacteria that the stringent response is linked to nutrient availability, we tested the growth of the \(\Delta\)_rsh_ strain in Plommet minimal medium, supplemented with erythroid as a carbon source, to mimic starvation conditions. The \(\Delta\)_rsh_ showed a clear growth defect compared to the WT as the OD rapidly decreased during the mid-exponential phase, indicating that \(\Delta\)_rsh_ cannot grow and survive in this medium, as expected for a mutant unable to produce (p)ppGpp (Figure 1B).  
Figure 1: Growth of the \(\Delta\)_rsh_ mutant in 2 YT rich medium (**A**) and in Plommet erythroid minimal medium (**B**). Strains were grown in liquid culture overnight in order to reach exponential phase. Cultures were then diluted at an optical density (OD) of 0.1 in 2 YT medium. The OD of each strain was measured every 30 min. The graph represents the means of a biological quadruplicate. The error bars represent the standard deviation for each time point. WT: wild type.  
We tested the ability of \(\Delta rsh\) to infect and multiply inside RAW 264.7 macrophages compared to the WT strain and the complemented strain by performing CFU counting throughout the cellular infection (Figure 3). The \(\Delta rsh\) mutant showed a significant decrease in CFUs at 24 h post-infection compared to the WT strain, suggesting that the _rsh_ gene is required for intracellular proliferation. A slight but significant difference was also observed at 2 h post-infection between the WT and complemented strain (Figure 3), probably highlighting a low toxicity of the vector or a _rsh_ overexpression effect.  
Figure 3: Survival and growth of _B. abortus_ WT, \(\Delta rsh\) and \(\Delta rsh\) pBBR-_rsh_ during infection of RAW 264.7 macrophages. Strains were grown in liquid culture overnight in order to reach exponential phase. Cultures were then diluted in Dulbecco’s Modified Eagle’s Medium (DMEM) to obtain a multiplicity of infection (MOI) of 50. The numbers of live bacteria (log\({}_{10}\)CFUs mL\({}^{-1}\) of cellular lysate, 0.5 mL per well) were determined at 2 h, 4 h, and 24 h by plating serial dilutions. Values represent the means of three independent experiments and the error bars represent the standard deviations. A one-way ANOVA test was performed as statistical analysis. The asterisks mean significant for \(p<0.05\) (*); \(p<0.001\) (***); \(p<0.0001\) (***).  
Figure 2: Survival and growth of _B. abortus_ WT, \(\Delta rsh\) and \(\Delta rsh\) pBBR-_rsh_ in 2 YT rich medium. Strains were grown in liquid culture overnight in order to reach exponential phase. Cultures were then diluted at an OD of 0.1 (\(3\times 10^{8}\) bacteria/mL for the WT strain) in 2 YT medium. The numbers of live bacteria (log\({}_{10}\)CFUs mL\({}^{-1}\)) were determined at 0 h, 4 h, 24 h and 48 h by plating serial dilutions. Values represent the means of three independent experiments and the error bars represent the standard deviation. The asterisks mean significant for \(p<0.05\) (*) \(p<0.01\) (**); \(p<0.0001\) (***), and the \(p\) values were calculated by one-way ANOVA.