## 2 Results and Discussion  
### Sequence Analysis of Rsh\({}_{\text{US6-1}}\) Protein  
The open reading frame of the full-length _rsh\({}_{\text{US6-1}}\)_ gene (accession number WP_007011921) was 2094 nucleotides in length. Rsh\({}_{\text{US6-1}}\) contained the nitrogen-terminal metal-dependent hydrolase domain (HD) (amino acids 26-177) and synthetase domain (Syn) (amino acids 236-347). The carboxy-terminal domain of Rsh\({}_{\text{US6-1}}\) consisted of the TGS domain (for: Thr-tRNA synthetase, GTPase and SpoT) (amino acids 385-444) and ACT domain (for: aspartate kinase, chorismate mutase and T protein) (amino acids 625-687) (Figure 1a). These four domains are commonly present in Rsh [3].  
Rsh\({}_{\text{US6-1}}\) displayed high sequence similarity to Rsh proteins whose functions had been identified in other strains [32; 33; 34; 35; 36]. The identities of Rsh\({}_{\text{US6-1}}\) with Rsh\({}_{\text{R}\text{r2-17}}\), Rsh\({}_{\text{Mtb}}\), Rsh\({}_{\text{Seq}}\), Rsh\({}_{\text{Ret}}\), SpoT\({}_{\text{Ecoli}}\) and RelA\({}_{\text{Ecoli}}\) were 88%, 37%, 38%, 43%, 38% and 30% respectively. In the HD domain (Figure 1b), Rsh\({}_{\text{US6-1}}\) contained six motifs, which are predicted to be essential for (p)ppGpp hydrolysis [26]. RelA\({}_{\text{Ecoli}}\) was more divergent in these motifs, which was consistent with its loss of hydrolase activity. In the Syn domain (Figure 1b), Rsh\({}_{\text{US6-1}}\) also contained five conserved motifs that were proved structurally and biochemically important for (p)ppGpp synthetase activity [26].  
The phylogenic analysis of RelA/SpoT homologs was performed (Figure 1c). The result showed that there was a clear separation of the RelA/SpoT families, forming five clusters: C1 for Rsh in _Alphaproteobacteria bacteria_, C2 for SpoT in _Gammaproteobacteria_, C3 for Rsh in _Firmicutes_. Rsh\({}_{\text{US6-1}}\) was grouped with Rsh\({}_{\text{R}\text{r2-17}}\) in C1. These  
Figure 1: Sequence analysis of Rsh\({}_{\text{US6-1}}\) protein. (**a**) Rsh\({}_{\text{US6-1}}\) domain; (**b**) amino acid alignment of RelA/SpoT homologs. Line 1: Rsh\({}_{\text{US6-1}}\). Line 2: Rsh\({}_{\text{R}\text{r2-17}}\). Line 3: Rsh\({}_{\text{Mtb}}\). Line 4: Rsh\({}_{\text{Seq}}\). Line 5: Rsh\({}_{\text{Ret}}\). Line 6: SpoT\({}_{\text{Ecoli}}\). Line 7: RelA\({}_{\text{Ecoli}}\) (see in Materials and Methods). Light grey boxes represent six conserved motifs in HD domain (HD 1: R. HD 2: H. HD 3: HD. HD 4: ED. HD 5: DR. HD 6: N) while dark grey boxes represent five conserved motifs in Syn domain (Syn 1: RKxoxScoxK. Syn 2: DxoxoK. Syn 3: YxxxH. Syn 4: ExQIRT. Syn 5: H) [26]; (**c**) phylogenetic tree based on RelA/SpoT homologs. Sequences, available at NCBI GenBank, were aligned by using the ClustalW algorithm [27] in MEGA version 6.0 [28]. Then a phylogenetic tree was constructed by the neighbor-joining method [29] with 1000 bootstrap replicates [30] after cutting off the redundant sequences at the end, with the use of MEGA version 6.0. The Rsh protein from _Anabena sp_. PCC 7120 (accession number BAB77915) was used as outgroup [31]. There were five clusters: C1 for Rsh in _Alphaproteobacteria_, C2 for SpoT in _Gammaproteobacteria_, C3 for Rsh in _Actinobacteria_, C4 for RelA in _Gammaproteobacteria_, C5 for Rsh in _Firmicutes_.  
data suggested that Rsh\({}_{\text{US6-1}}\) possessed the sequence required for the (p)ppGpp synthetase and hydrolase activities.