## 4 Potential Mechanism of Virulence  
In _E. coli_ and _Salmonella_, it has been shown that GidA and MnmE bind together to modify tRNA using a post-transcriptional mechanism in which both proteins are interdependent [16, 21, 22, 30]. GidA and MnmE were first identified as potential tRNA modification enzymes during a screen for novel _E. coli_ mutants defective in mmm\({}^{5}\)s\({}^{2}\)U biosynthesis [21]. Crystal structures of MnmE show a dimeric protein with each monomer consisting of three domains which are the _N_-terminal, central helical, and GTP-binding domains [69, 70]. The crystal structure of GidA reveals a FAD-binding site and dimer interface [24, 25, 71]. Each GidA homodimer subunit consists of three domains which are the first \(\alpha\)/\(\beta\) domain (also called the FAD-binding domain), a second \(\alpha\)/\(\beta\) domain, and a \(\alpha\)-helical \(C\)-terminal domain [24, 25, 71]. The mechanism by which GidA and MnmE form a heterotetrmeric complex is not fully understood, but it has been shown that the \(\alpha\)-helical \(C\)-terminal domain of GidA along with the two-fold symmetry \(N\)-terminal domain of MnmE are required for GidA/MnmE interaction [24, 25, 71]. Once the GidA/MnmE interaction occurs, the complex catalyzes two different GTP-and FAD-dependent reactions which results in 5-aminomethyluridine (nm\({}^{5}\)U) and 5-carboxymethylaminomethyluridine (cmmm\({}^{5}\)U) by using ammonium and glycine, respectively, as substrates [23]. GTP hydrolysis by the MnmE G-domain leads to structural rearrangements of the GidA/MnmE complex which has been shown to be essential for tRNA modification [15, 72]. More work is needed, however, to determine the mechanism of tRNA recognition and substrate selection by the GidA/MnmE complex. The GidA/MnmE pathway has been implicated as a major pathogenic regulatory mechanism in bacteria as evidenced by the numerous pathogens that have been attenuated following deletion of _gidA_ and/or _mmmE_. The mechanism by which this pathway regulates bacterial virulence is unclear. It has been shown that deletion of _gidA_ and/or _mmmE_ increases the occurrence of a two-base frameshift during the translation of mRNA [22]. This hypothesis is plausible as it is well known that modifications at the wobble position of the anticodon are needed to accurately and efficiently synthesize proteins [73, 74]. Furthermore, alterations in the modification levels of the wobble position can affect the synthesis of specific proteins [73, 75, 76, 77]. This mechanism of translational fidelity suggests a role of global regulation by GidA and MnmE as evidenced by their mutations affecting numerous phenotypic traits. Despite the conservation of GidA and MnmE among different bacteria, the phenotypes of different _gidA_ and/or _mmmE_ mutants do not suggest a common mechanism for the functions of these enzymes in bacterial pathogens. This could be due to the prevention of frameshifting of genes encoding virulence proteins in the different organisms since base modification contributes to the efficiency and accuracy of translation [3, 22, 78]. Also, studies of GidA have been complicated because homologues of GidA exist in two sizes, and are transcribed from genes that are not genetically linked [10]. For example, the study by [PERSON] _et al._ shows that deletion of _gidA_ results in a nearly normal transcriptional profile with an alteration of several virulence proteins. Additionally, their study showed deletion of _mmmE_ resulted in nearly the same phenotypes as deletion of _gidA_[37]. In contrast, studies in _Salmonella_ show that deletion of _gidA_ results in a down-regulation of both virulence genes and proteins, and although deletion of _mmmE_ results in the same attenuated phenotypes as a _gidA_ deletion mutant, a _gidA_ deletion mutant appears to be more attenuated than a _mmmE_ deletion mutant [30, 38]. One plausible explanation for this could be their location in the chromosome.