# Regulation of the _Pseudomonas syringae_ Type III Secretion System by Host Environment Signals
## 3 Molecular Mechanisms of T3 SS Deployment in Response to Environmental Stimuli
### Negative Regulation of the T3 SS by the GacSA Global Regulatory System  
The GacSA two-component system is conserved throughout Pseudomonas spp. and has been broadly characterized for its pleiotropic roles as a regulator of secondary metabolism and host-microbe interactions [122]. In this two-component system, the inner membrane-localized sensor kinase GacS responds to an unknown stimulus and activates cytoplasmic response regulator GacA by phosphorelay [123; 124; 125; 126]. The GacSA system was initially identified as a regulator of _P. syringae_ virulence over 30 years ago. In this work, isolates of _P. syringae_ pv. _syngiae_ B728a containing insertion mutations in _gacS_ were identified for their inability to cause disease lesions on host bean plants [127]. Based on these observations, the GacS sensor kinase in _P. syringae_ was originally named Lema (for Lesion Manifestation \(\underline{\Delta}\)). The authors were unable to identify a cognate response regulator in the genomic neighborhood of _gacS_, despite two-component systems being typically encoded together within operons in bacterial genomes [128; 129; 127]. GacS was later genetically determined to form a two-component system in _P. syringae_ with FixJ-like response regulator GacA, similar to the BarA/UvrY two-component system in _Escherichia coli_[123; 125; 130]. Throughout the \(\gamma\)-proteobacteria, _gacA_ is located distally from _gacS_ and shares an operon with _uvrC_, encoding a component of the nucleotide excision repair complex, though the functional significance of this pairing is unclear [122; 131]. The role of GacSA as a regulator of the T3 SS in _P. syringae_ has been clouded by conflicting experimental results. While some studies concluded that GacSA positively regulates T3 SS expression in _P. syringae_[63; 95; 132], other studies reported that GacSA negatively regulated T3 SS expression, and was dispensable for T3 SS deployment in the plant environment [127; 133]. In contrast to conflicting _P. syringae_ studies, GacSA was identified as a negative regulator of the T3 SS in the animal pathogen _Pseudomonas aeruginosa_[134; 135; 136]. The ambiguous role of GacSA in _P. syringae_ has complicated the development of cohesive models of GacSA function and T3 SS regulation among all _Pseudomonas_ pathogens. An isolate of _P. syringae_ pv. tomato DC3000 containing a Tn5::_gacA_ insertion, termed AC811, was studied to determine the molecular basis of GacSA regulation of virulence [63]. The authors demonstrated that the AC811 mutant was attenuated in both virulence and production of the T3 SS in the plant environment. Largely based on this study, the role of GacSA as a regulator of virulence through positive control of the T3 SS became the prevailing model in _P. syringae_, reflected in various contemporary reviews [137; 138; 115] and mathematical models of virulence regulatory networks in _P. syringae_[139]. Contrary to this early model, evidence has recently emerged to suggest that GacSA functions as a negative regulator of T3 SS in _P. syringae_. A re-evaluation of the DC3000 gacA_::Tn5 isolate AC811 and other DC3000 _gacA_: loss-of-function mutants revealed that the inactivation of _gacA_ enhanced, rather than reduced, T3 SS gene expression. Furthermore, this study demonstrated that _gacA_ was not required for _P. syringae_ T3 SS deployment and virulence within the host foliar apoplast [140]. Rather, attenuated virulence of the _gacA_::Tn5 AC811 strain was attributed to a second-site mutation in _annK_, an enzyme involved in bacterial cell wall recycling, as well as negative polar effects of the _gacA_::Tn5 insertion on the transcription of downstream gene _uvrC_[141]. While neither _annK_ nor _uvrC_ were previously associated with _P. syringae_ virulence, a polar effect of a _gacA_ mutation on the expression of downstream _uvrC_ was reported to impact the virulence of the closely related animal pathogen _Pseudomonas aeruginosa_[142; 143].