## 2 Results
### The Artificial Hydrolysis of (p)ppGpp Leads to a \(\Delta\)rsh Phenotype during Infection  
Since Rsh is responsible for (p)ppGpp homeostasis and an \(\Delta\)_rsh_ mutant failed to proliferate inside RAW 264.7 cells, we tested the involvement of (p)ppGpp in the infection process. However, it is known that Rsh is involved in regulation networks through protein-protein contacts [14; 24] in other bacteria. Therefore, we cannot rule out that the absence of the Rsh protein, rather than the absence of (p)ppGpp, would be responsible for the defect observed in infection. This is reinforced by the observation that mutants for homologs of the glutamine-dependent control pathway of Rsh are also attenuated in RAW 264.7 macrophages [25]. We thus generated a strain in which (p)ppGpp is hydrolyzed by a strong (p)ppGpp hydrolase, a product of the _mesh1_ gene from _Drosophila melanogaster_[26]. Indeed, it was shown that Mesh1 was active in vitro and in vivo [26]. We thus expect this heterologous enzyme to be constitutive in _B. abortus_. We adapted the _mesh1_ coding sequence for the codon bias of _B. abortus_ and expressed the resulting coding sequence on a medium copy replicative plasmid, leading to the _B. abortus_ pBBRi-_mesh1b_ strain. Interestingly, this strain showed a clear decrease in CFUs at 24 h post-infection of RAW 264.7 macrophages (Figure 4), which is consistent with a crucial role played by (p)ppGpp to allow growth inside host cells, as suggested above. Expression of a Constitutive Allele for a (p)ppGpp Synthetase Impacts Bacterial Growth and Chromosome Replication  
In order to get more insight about the role of (p)ppGpp in _B. abortus_, we constructed a strain that artificially produces this alarmone. We used a truncated version of the _relA_ gene from _E. coli_, _relA'_[12] that removes the C-terminal regulatory domains of the encoded protein. The _relA'_ coding sequence was inserted downstream of an isopropyl \(\beta\)-D-1-thiogalactoside (IPTG)-inducible promoter on the pSRK replicative plasmid [27]. The resulting strain, named _pSRK-relA'_, is supposed to produce (p)ppGpp synthetase when IPTG is added to the medium. As a negative control, we used the _pSRK-relA''_ strain containing the point mutation E335Q, which leads to a catalytically dead protein. Since the detection of (p)ppGpp levels using \({}^{32}\)P is not compatible with our biosafety level 3 set up, we tried to gain indirect  
Figure 4: Survival and growth of _B. abortus_ WT and pBBRi-_mesh1b_ 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 DMEM to obtain a MOI of 50. The numbers of live bacteria (log\({}_{10}\)CFUs mL\({}^{-1}\)) were determined at 2 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 \(t\) test was performed for the comparison of the two strains. The asterisks mean significant for \(p<0.01\) (**) and “ns” means “not significant”. evidence that (p)ppGpp is indeed produced when the expression of _relA'_ is induced. We assayed the growth of the _pSRK-relA'_ and _pSRK-relA'_* strains in rich culture medium with or without IPTG induction. The _pSRK-relA'_, _pSRK-relA'_* and WT strains grew equally in 2 YT; however, when IPTG was added to the medium, a growth delay was only observed for the _pSRK-relA'_ strain (Figure 5). This observation is consistent with the production of (p)ppGpp levels that are sufficient to limit growth when _relA'_ expression is induced. Since it was already reported that (p)ppGpp has an impact on DNA replication in _C. crescentus_ and _E. coli_[12; 13; 14; 15], we took advantage of a _B. abortus_ strain allowing us to monitor the chromosomal replication status at the single cell level in order to study the impact of (p)ppGpp overproduction on DNA replication.