## 3 Discussion  
_Brucella abortus_ is able to control its cell cycle progression when it is inside host cells, particularly the replication and segregation of its replication origins [2]. However, the molecular mechanisms involved in this control are unknown. Since convincing data show that the ability to adapt to starvation is a key factor for the success of cellular infections by _Brucella melitensis_ and _Brucella suis_[10; 11],  
Figure 7: Survival of _B. abortus_ WT and _pSRK-reIA_’ strains with and without IPTG during infection of RAW 264.7 macrophages. Strains were grown in liquid culture overnight in order to reach exponential phase. Cultures were then diluted at an OD of 0.1 with or without IPTG (1 mM) and were incubated for 3 h at 37 \({}^{\circ}\)C. Cultures were then diluted in DMEM with or without IPTG (10 mM) to obtain a MOI of 50. Concentrations of live bacteria (log\({}_{10}\)CFUs mL\({}^{-1}\)) were determined at 0 h, 4 h, and 24 h post-infection by plating serial dilutions. Values represent the means of three independent experiments and the error bars represent the standard deviation. A Student’s t test was performed as statistical analysis. The asterisks mean significant for \(p<0.05\) (*) and \"ns” means ”not significant”. we further investigate the role of the (p)ppGpp, the alarmone produced in the presence of starvation conditions, and the Rsh enzyme that is proposed to synthesize this alarmone. We first confirmed that in _B. abortus_ 544, like in other _Brucella_ strains, Rsh is crucial for the success of a cellular infection (Figure 3), for the survival in stationary phase (Figure 2) and the growth in minimal medium (Figure 1). In agreement with the absence of proliferation of the _rsh_ mutant in macrophages, a strain constitutively producing a (p)ppGpp hydrolase (Mesh1b) from _Drosophila melanogaster_ is also unable to grow in RAW 264.7 macrophages. We observed that the survival of _pBBR-mesh1b_ strain is less severely impacted during infection than the _\(\Delta\)rsh_ strain. One could imagine that this intermediate phenotype is due to the presence of residual alarmone in the _pBBR-mesh1b_ strain, while it is not the case in the _\(\Delta\)rsh_ strain since the (p)ppGpp synthetase domain is not present. Another explanation for these different phenotypes could be that Rsh plays additional role(s) for survival in infection than the regulation of (p)ppGpp homeostasis. Interestingly, a mutant overproducing (p)ppGpp is also unable to proliferate in these cells, suggesting that the (p)ppGpp level should be in a specific range of concentration to allow cellular infection; having too much or not enough (p)ppGpp would be detrimental for the success of the cellular infection. What does (p)ppGpp control and how is Rsh regulated? It was shown that the absence of _rsh_ in _B. suis_ affects the transcription of genes known to be involved in virulence [28], such as _pyrB_, which was shown to be essential for _B. abortus_ proliferation in RAW 264.7 macrophages [25]. It is thus likely that a minimum level of (p)ppGpp would be required for the success of a cellular infection by _B. abortus_. It was shown that the glutamine pool modulates Rsh (called SpoT) in the model alpha-proteobacterium _Caulobacter crescentus_ through the phosphotransferase system (PTS) [14]. Interestingly, mutants for components of this system were found to be attenuated in RAW 264.7 macrophages [25]. Moreover, PTS and the two-component regulator BvrR control the expression of the _virB_ operon [29; 30], coding for a type IV secretion system that is crucial for intracellular proliferation in most cell types [31]. These data indicate that a quite complex regulation network is probably linking Rsh control and virulence. However, the molecular mechanisms controlling Rsh activity in _B. abortus_ are unknown and deserve further investigation. One striking conclusion of our data is the moderate effect that (p)ppGpp overproduction has on the proportion of bacteria at the G1 stage of the cell cycle.