## 2 Results
### The Artificial Hydrolysis of (p)ppGpp Leads to a \(\Delta\)rsh Phenotype during Infection
This strain expresses an _mCherry-parB_ allele that allows us to highlight the segregation of replication origin(s) of chromosome I. In this strain, one mCherry focus means that segregation has not yet started and the bacterium is probably in the G1 phase of the cell cycle, and two mCherry foci correspond to two segregated replication origins, meaning that the bacterium has already started replication and is thus in the S or G2 phase of the cell cycle [2]. The _pSRK-relA'_ and _pSRK-relA'_* plasmids were inserted in a _B. abortus mCherry-parB_ strain and we counted the number of G1 bacteria every two hours for 6 h after the inoculation of bacteria in rich medium, with or without IPTG. Interestingly, we observed an increase in the proportion of G1 bacteria over the time of induction with IPTG for the _pSRK-relA'_ strain (Supplementary Figure S3). The proportion of G1 bacteria of the non-induced _pSRK-relA'_ and both the induced or non-induced _pSRK-relA'_* remained stable after the addition of IPTG (Figure 6). These results strongly suggested that artificial induction of (p)ppGpp synthesis could delay the transition between the G1 phase to the S phase and subsequently have an impact on the initiation of chromosomal replication in _B. abortus_. Figure 5: Growth curve in rich culture medium for the WT, _pSRK-relA’_ and _pSRK-relA’_* with or without IPTG. Strains were grown in liquid culture (2 YT medium) overnight in order to reach exponential phase. Cultures were then diluted at an OD of 0.1 in 2 YT medium supplemented or not with IPTG. The OD of each strain was measured every 30 min. The graph represents the means of a biological triplicate. The error bars represent the standard deviation for 3 biological replicates for each time point. Figure 6: Proportion of G1 bacteria in rich culture medium with or without IPTG for the _PSRK-relA’_ and _pSRK-relA*_ strains. (**A**) Schematic drawing of the mCherry-ParB localization throughout the cell cycle [2] and fluorescence microscopy of the _pSRK-relA’mCherry-parB_ strain. Scale bar represents 5 μm. (**B**) Strains were grown in liquid culture (2 YT medium) overnight in order to reach exponential phase. Cultures were then diluted to an OD of 0.1 in 2 YT medium supplemented or not with IPTG. Samples were taken every 2 h, placed on a phosphate-buffered saline (PBS) agarose pad and observed with a fluorescence microscope. Bacteria in G1 phase (presenting only one focus of mCherry-ParB) were counted for each time post-induction. Error bars represent the standard deviation from the means of three independent experiments (biological triplicates). The significant differences are indicated by \(p\) < 0.05 (*), \(p\) < 0.01 (**) and \(p\) < 0.001 (**); “ns” means not significant. The number of bacteria considered in these triplicate experiments are detailed in Supplementary Table S1. Induced Production of a Constitutive (p)ppGpp Synthease Leads to a Proliferation Defect during Infection  
Since (p)ppGpp overproduction seemed to have an impact on replication, i.e., an increase of the proportion of G1 cells in the bacterial population, and that the G1 bacteria are more infectious, we decided to investigate the effect of overproduction of (p)ppGpp on the infection process. We infected RAW 264.7 macrophages with the _pSRK-reIA_' strain induced or not with IPTG (Figure 7). The IPTG was kept in the cell culture medium during the infection for the induced condition. We first observed that bacterial internalization is not enhanced by the increase in the proportion of bacteria in the G1 phase of the cell cycle. We also observed that induction of _pSRK-reIA_' induced a strong defect in intracellular proliferation compared to the WT and uninduced _pSRK-reIA_' conditions. This result suggests that overproduction of (p)ppGpp during infection prevents growth in the intracellular niche.